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Concentration-Gradient Microfluidic Chips for Drug Screening

Fangjuan Zhang , Haibing Liu , Mengqi Gao , Defu Wang , Yanbing Niu , Shaofei Shen

Progress in Chemistry ›› 2021, Vol. 33 ›› Issue (7) : 1138 -1151.

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Progress in Chemistry ›› 2021, Vol. 33 ›› Issue (7) : 1138-1151 DOI: 10.7536/PC200717
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Concentration-Gradient Microfluidic Chips for Drug Screening
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Abstract

Microfluidic chip is one of the most representative technologies of micro-analysis platform in this century. It has many advantages such as low reagent consumption, analytical miniaturization, easy control and integration, high automation and good biological compatibility. It has outstanding performances in many fields such as biology, medicine, food and environment, especially in the field of drug screening which has achieved a series of achievements with its own advantages. This article summarizes the research progress of concentration gradient microfluidic chips for drug screening in recent years, including paper chip, hydrogel chip, droplet chip and polydimethylsiloxane (PDMS) chip. Furthermore, the advantages and disadvantages of concentration gradient microfluidic chip in single cell analysis, combination drug screening, three-dimensional (3D) cell culture, cell microenvironment simulation are described. Finally, we provide a perspective for its application prospects.

Contents

1 Introduction

2 Characteristics of concentration-gradient microfluidic chip

3 Type of concentration-gradient microfluidic chip

3.1 Paper chip

3.2 Hydrogel chip

3.3 Droplet chip

3.4 PDMS chip

4 Application of concentration-gradient microfluidic chip

4.1 Single cell analysis

4.2 Combination drug screening

4.3 3D cell cultures

4.4 Simulated cell microenvironment

5 Conclusion and outlook

Graphical abstract

Keywords

microfluidic chip / concentration gradient / drug screening

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Fangjuan Zhang, Haibing Liu, Mengqi Gao, Defu Wang, Yanbing Niu, Shaofei Shen. Concentration-Gradient Microfluidic Chips for Drug Screening. Progress in Chemistry, 2021, 33(7): 1138-1151 DOI:10.7536/PC200717

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1 Introduction

Gene sequencing and proteome sequencing technologies have opened new doors to understanding various biological processes and disease mechanisms[1]. The rapid development of drug development can effectively help people prevent infection and treat diseases, and at the same time, it is conducive to the substantial reduction of medical costs[2]. However, in the field of drug screening, traditional methods are facing many challenges. Traditional drug screening is mainly carried out through multi-well plates and Petri dishes, which consume too much time and labor costs. At the same time, because cell culture is carried out in a semi-static environment, it can not accurately simulate the cell microenvironment. Animal models play a vital role in drug screening and development, but the scientific community is reducing the use of animals in experiments because of ethical concerns. More seriously, due to platform and other constraints, the failure of screened candidate drugs in clinical trials has greatly reduced the efficiency of drug development. Therefore, the research on efficient drug screening platform has attracted much attention.
Microfluidic chip technology is a precise manipulation technology of microfluidic interface, which was proposed by Swiss scientists Manz and Widmer in the early 1990s. It is also one of the most representative micro-analytical technology platforms in the 21st century[3]. Compared with traditional analytical techniques, microfluidic chip is a "micro" and "complete" analytical technology platform, and it has the advantages of simple operation, time saving and high efficiency. In recent years, as the focus of the development of micro total analysis system, the application of microfluidic chip technology has involved many interdisciplinary research fields, such as biological/medical research, biological simulation, organic synthesis, disease diagnosis, drug screening and environmental monitoring[4⇓~6]. In the research system of microfluidic chip, the concentration gradient microfluidic chip is one of the most important components in biological research. The concentration gradient microfluidic chip can simultaneously generate various concentration gradients without manual pipetting to construct the concentration gradients[7]. Drug screening and therapy optimization usually need to study the dose-dependent cellular response under different drug concentrations, so the concentration gradient microfluidic chip has become a powerful tool in this field. Concentration gradient microfluidic chips can quantitatively and massively evaluate the toxicity and optimal concentration of different drugs, which can not only improve throughput and reduce experimental costs, but also quickly and accurately control and interact with each other, with higher gradient resolution.
Based on our previous research experience in the field of microfluidic chips, this paper reviews the latest research progress in the field of drug screening based on concentration gradient microfluidic chips.Different types of concentration gradient microfluidic chips, including paper-based microfluidic chip, hydrogel microfluidic chip, droplet microfluidic chip and polydimethylsiloxane (PDMS) microfluidic chips, were described.At the same time, the applications of the concentration gradient microfluidic chip in single cell analysis, combinatorial drug screening, three-dimensional (3D) cell culture and simulation of cell microenvironment were described, and the development prospects of the concentration gradient microfluidic chip were prospected[8⇓⇓⇓⇓⇓⇓⇓~16].

2 Characteristic of concentration gradient microfluidic chip

Traditional drug concentration gradient experiments are mainly carried out on multi-well plates, which not only requires tedious and repetitive steps, but also requires excessive time and energy. With the progress of microfluidic technology and the wide application of microfluidic chips in biological research, concentration gradient generators based on microfluidic chips are developing prosperously.The concentration gradient microfluidic chip has attracted much attention because of its advantages of low reagent consumption, easy control and automation, and has been widely used in the field of drug screening[17⇓⇓~20]. At the same time, compared with macro-scale systems, the micro-scale characteristics of microfluidic devices can effectively reduce the use of chemical reagents and expensive drugs, and can produce precise concentration gradients by providing flexible drug concentration adjustment. In addition, a series of concentrations can be produced simultaneously in different reaction chambers in a system, so that biological and chemical reactions can be carried out simultaneously at different drug concentrations, in which case each reaction chamber is a research system capable of simultaneously studying the effects of different concentrations of drugs on cells[21,22]. The gradient obtained by the concentration gradient microfluidic chip is predictable and repeatable while being controllable and quantitative. In addition, concentration gradient microfluidic chips have shown great advantages in speed, cost, sample/reagent consumption, contamination, efficiency and automation, and have a wide range of applications.

3 Concentration gradient microfluidic chip type

3.1 Paper-based microfluidic chip

Paper-based microfluidic chip devices are one of the most promising point-of-care test platforms.Compared with lithography-based microfluidic chips, paper-based microfluidic chip devices have the advantages of portability, absorbability, disposability and low cost, and do not need to use precision pumps or valves like traditional liquid devices during operation[23]. In addition, paper is a porous matrix made of cellulose, which is excellent in liquid absorption, and when certain areas of the paper are Hydrophobically modified, the aqueous solution applied to the paper will be precisely guided through the hydrophilic areas by capillary effect. Paper-based microfluidic chip devices have broad application prospects in portable and low-cost analysis, especially in personalized health care, nursing diagnosis and drug screening based on bioassay[24].
Liu et al. Developed a simple 3D printing paper-based microfluidic platform[25]. The platform designs a "Christmas tree" structure on the top and bottom of the chip device, which can continuously generate concentration gradients, and a paper-based chip cell culture area for drug screening research. As shown in fig. 1A, the two-layer structure separates the concentration gradient forming layer from the cell culture layer, which simplifies the culture of cells in the microfluidic chip, and the scaffold can protect cells from the shear force of continuous liquid flow, which is convenient for cell culture and subsequent detection. Subsequently, the effect of H2S on human liver cancer cells (SMMC-7721) was studied through the paper-based microfluidic platform. The results showed that after entering the cancer cells, the H2S interacted with the nitric oxide in the cells to form intermediate polysulfides with biological activity, which caused a series of physiological changes in the cancer cells and eventually led to apoptosis. This new 3D printing paper-based microfluidic platform has a wide range of applications in cell research and drug screening. As shown in fig. 1B, Wu et al. Proposed a simple, low-cost paper-based microfluidic chip continuous perfusion device (μPAD)[26]. The platform is mainly composed of a liquid supply device and an absorption device. Human breast cancer cells (MDA-MB-231) were inoculated into 9 cell culture areas of the μPAD, and paclitaxel solution was infused into the μPAD through the infusion device. Different concentrations of paclitaxel solution could be formed in one μPAD at the same time by using the continuous infusion device, which acted on the cells in the cell culture area. The results showed that the viability of cells treated with different concentrations of paclitaxel was significantly different. As a lightweight and biofluid-based microfluidic platform, the device can be applied to biological and medical fields such as cell culture and drug screening.
In addition, Hong et al. Designed a paper-based concentration gradient generator to generate a series of different concentrations of doxorubicin through paper fiber diffusion, which acted on human cervical cancer cells (HeLa) cultured in microarrays[27]. The results showed that after the HeLa cells were cultured in the drug for 8 H, the inhibition efficiency of the drug was positively correlated with the concentration of the drug. This device, which combines a paper-based microfluidic chip and a cell culture array, shows potential for drug development and high-throughput drug screening. Chen et al. Designed a paper-based microfluidic chip device for cell culture and drug screening. Human liver cancer cells (HepG2) were cultured on the paper-based microfluidic chip for 24 H, and then the adhered HepG2 cells were incubated with different concentrations of cyclophosphamide for 24 H[28]. The data showed that the cell death rate increased as the drug concentration increased. The results indicate that the paper chip device can be used as an efficient, inexpensive and high-throughput cellular drug screening tool. Ge et al. Developed a simple high-throughput drug screening platform by designing micropores on paper substrates[29]. In order to verify the feasibility of drug screening, two kinds of human liver cancer cells (Huh7 and HepG2) and two kinds of drugs (doxorubicin and etoposide) were selected. Huh7 cells and HepG2 cells were cultured in paper matrices and treated with different concentrations of doxorubicin and etoposide, and the dose-response curves of different cell lines and drug combinations were plotted. The results showed that the drug resistance rate of Huh7 cells was higher than that of HepG2 cells, and the efficacy of adriamycin in the treatment of liver cancer was better than that of etoposide. The device provides a quantitative, simple and high-throughput platform for cell drug sensitivity research and drug screening.
As one of the cheapest materials for microfluidic chip design, paper has low manufacturing cost and is easy to stack to form multi-layer microfluidic channels. However, there are also challenges, because paper has low surface tension, liquid can not be well confined in hydrophobic channels, and liquid in open channels is easy to volatilize, resulting in leakage of samples and reduced utilization of samples. At the same time, the detection sensitivity of paper-based microfluidic chips is often not very high, which makes it impossible to achieve high-precision detection.

3.2 Hydrogel microfluidic chip

The cellular microenvironment consists of extracellular matrix (ECM), adjacent cells, and soluble factors. The ECM provides mechanical support and chemical stimulation to cells, and guides cell proliferation, migration, differentiation and apoptosis. The ECM also contains soluble macromolecules, such as growth factors, chemokines and cytokines[30,31]. Therefore, it is very important to replicate the cell microenvironment in vivo when constructing the gradient generator in vitro. Hydrogel matrix has porosity and pore size controllability, which can simulate the characteristics of extracellular matrix, effectively absorb water during cell culture, and is permeable to gases, nutrients, proteins and signaling molecules[32,33]. Microfluidic gradient generators based on hydrogels provide a higher level of precision, quantification, and spatiotemporal gradient control than traditional methods, which can greatly improve the understanding of a variety of biological phenomena.
Extracellular matrix not only supports the structure of cells, but also is important for cell guidance in different biological processes such as embryonic development, adulthood, wound healing and tissue repair. The extracellular matrix is composed of scaffold macromolecules embedded with soluble and matrix-bound bioactive molecules (growth factors, cytokines, hormones, nutrients, gases, etc.), so the realization of soluble or covalently attached cell-targeting cues in biomaterials poses a great challenge to the research and production of biomaterials.Hydrogels, on the other hand, are considered to be synthetic substitutes for natural extracellular matrices because of their key functions such as 3D solid support, fibrous structure, gas and nutrient permeability, and allow the storage and release of bioactive molecules. As shown in fig. 2 (a), the hydrogel 3D gradient can provide soluble guidance cues, allowing cells to be cultured on top of or within the 3D hydrogel matrix, and the hydrogel matrix contains covalently immobilized growth factor gradients or specific adhesion sequences to guide cells, facilitating the study of cell alignment and migration in a 3D environment[34]. Ladet et al. Proposed a processing method of physical hydrogel materials based on amphiphilic polymers. Chitosan is a natural amphiphilic copolymer with biodegradability, bioactivity and biocompatibility.The relative proportions of acetylated and deacetylated residues play an important role in the balance between hydrophilic and hydrophobic interactions and in cell proliferation and tissue regeneration[35]. They proposed an onion-like multi-membrane biomaterial based on chitosan hydrogel, and studied the neutralization step of different concentrations of sodium hydroxide (NaOH) aqueous solution and the change of chitosan mass fraction with the concentration of NaOH in the neutralization solution during the gel neutralization process. This method greatly simplifies the processing of gels with complex shapes and multi-membrane structures, and has potential biomedical applications. In addition, He et al. Developed a fast and simple method for the generation of chemical concentration gradients in microfluidics based on hydrogels[36]. By integrating the portable microfluidic operation and the photopolymerization, the method can rapidly and simply generate a concentration gradient with continuous change, and the prepared hydrogel has certain cell adhesion and porosity, and is expected to become a powerful tool for wide biological and tissue engineering applications. As shown in fig. 2 (B), Hu et al. developed a concentration gradient hydrogel array microfluidic device for high-throughput drug screening[37]. Human monocytic leukemic cells (THP-1) were aggregated into hundreds of multicellular masses of controlled size. Instead of continuous bulk material, the photosensitive gelatin-methacryloyl (GelMA) hydrogel column array containing cell clusters under drug concentration gradient was obtained by selective area exposure method. The results showed that the cell aggregation activity was inversely proportional to the drug concentration in the hydrogel column, and the sensitivity to drug toxicity was lower than that of monolayer cultured cells. Three-dimensional multicellular arrays provide a number of in vitro tumor models that can be used directly for downstream drug testing. This technology inherits the advantages of acoustic assembly and has the advantages of more flexibility, practicality and high throughput. Mahadik et al. describe a method for repeatedly creating a heterogeneous microenvironment containing hematopoietic stem cells (HSCs) and one or more putative niche cells in a reverse gradient hydrogel based on a microfluid-to-fluid mixing platform[38]. As shown in Figure 2 (C), the method is inspired by HSC niches to create small-volume three-dimensional hydrogel structures containing overlapping patterns of cell and matrix components. The effectiveness of the multi-gradient hydrogel was verified at multiple scales (from single cells to bulk structures) using a variety of analytical methods. The local properties of these hydrogels at multiple scales were characterized by three different analytical methods: (1) bulk structural fluorescence analysis; (2) multiphoton imaging of a single cell within the structure; (3) extraction from discrete subregions of the hydrogel after incubation. The system creates a series of stable gradients in a single, optically translucent three-dimensional biomaterial, enabling research on how microenvironmental signals affect cell fate and optimizing the culture platform for cell expansion in vitro. Liu et al. Reported a microfluidic platform using a multidirectional hydrogel barrier to simultaneously establish multiple biochemical cue gradients for the study of axonal attraction and repulsion integration[39]. As shown in Figure 2 (d), the platform can be used for research and analysis of neural development, drug development and screening, damaged tissue repair and other aspects by performing on-chip bionic neuron culture and controlling multi-gradient concentration distribution to study the integration of axon attraction and repulsion.
Hydrogels are generally biocompatible, similar to extracellular matrix, in which microchannels can be established for solution delivery and cell culture. However, due to the inherent defects of low density and low strength of hydrogels, the resolution of hydrogels in micro-scale processing is lower than that of other polymers (nano-scale), which affects the precise construction of internal channels in chips. In addition, cell-encapsulated hydrogels are often incompatible with some microfabrication processes, which affects the fabrication of microdevices.

3.3 Droplet microfluidic chip

Droplet microfluidics has attracted attention in recent years because it provides a way to prepare monodisperse droplets that can be used as individual microreactors when chemical or biological reagents are added. At the same time, droplet microfluidic technology also has the advantages of compartmentalization, low contamination, low dispersion, rapid mixing, low reagent consumption and low cross-contamination, which shows great potential for analysis and detection. The advantages of micro-scale modular operation are conducive to sample preparation and separation. Through template operation, more analysis and detection can be better integrated[40,41].
Song et al. Proposed a unique microfluidic system that can control a variety of chemical reactions in the millisecond scale[42]. As shown in fig. 3 (a), it is possible to control when each reaction starts, when it develops before it is separated or combined with other reactions, and when each reaction is analyzed or terminated. The droplets formed within the microfluidic channel can act as a microreactor, reacting in separate flow droplets containing reagent A, reagent B, and containing buffer, and the droplets can be transported through the microchannel. The system realizes mixing and transmission in milliseconds, which is beneficial to the study of chemical analysis and synthesis as well as complex reaction networks. Rotman studied single enzyme kinetics in droplets by simply dispersing an extremely dilute aqueous solution of β-D-galactosidase into oil droplets and measuring the conversion with a fluorescence microscope[43]. Droplets formed using conventional techniques are far less uniform than those formed using microfluidic platforms. The microfluidic platform-based preparation of monodisperse droplet for single enzyme kinetic analysis can be used for experimental study of biology, chemistry and that like. In addition, as shown in Fig. 3 (B), Sun et al. proposed a gradient generation method based on a unique physical principle, which generates a concentration gradient in a long moving plug by continuously extracting the sample on the sheet, mixing the sample with water in the plug, and then breaking the gradient plug into chemically different droplets[44]. It can produce a moving droplet array that contains chemical concentration variations between individual droplets. The method has the following significant advantages: (1) the dilution range of the concentration can be adjusted by various control parameters (including the plug flow velocity, the volume of the fixed droplet and the moving plug), thereby being more flexible; (2) a plurality of dilution plugs can be provided in one step, thereby realizing unlimited dilution of the original sample; (3) High throughput and fine resolution, producing at least 6500 10 nL droplets per hour; (4) The ability to replace pre-stored sample drops and perform serial dilutions, thereby enabling the screening of multiple targets. The method has the capability of loading, replacing and diluting multiple samples on the chip, effectively realizes the rapid generation of concentration gradients and the dynamic control of gradient ranges, and can carry out high-throughput and high-resolution screening. Furthermore, Cai et al. described a microfluidic droplet based gradient generator for enzyme inhibition assay as shown in fig. 3 (C), which combines the flow injection gradient technology with the droplet microfluidic technology to generate a droplet array with a concentration gradient, and automatically injects a nanoscale sample solution into the chip in a valveless flow injection analysis mode[45]. More than 240 enzyme inhibition reactions with different inhibitor concentrations can be carried out in 2. 5 min. Compared with the multi-well plate screening system, the amount of inhibitor used is reduced by 1000 times, and the device generates large-scale concentration gradients with very low sample consumption, demonstrating its potential in high-throughput drug screening.
The main characteristic of droplet microfluidic chip is that it can produce monodisperse droplets. This monodisperse droplet can be used as a separate microreactor with the characteristics of small volume, low diffusion and low cross-contamination. However, the droplet microfluidic chip also has some shortcomings, such as the difficulty of operation, the instability of the droplets, the easy evaporation of aqueous droplets and the difficulty of droplet sorting.

3.4 PDMS microfluidic chip

Polymer chips were introduced a few years after silicon/glass chips. There are many kinds of polymer materials, which have great flexibility in choosing materials with specific properties. Compared with inorganic materials, polymer materials are easy to obtain and cheap, and have become the most commonly used microchip materials. The most popular elastomer in microfluidics is polydimethylsiloxane (PDMS), which has the advantages of easy microfabrication and low cost. In addition, PDMS has the properties of high elasticity and gas permeability, which are essential for long-term cell culture in sealed microchannels, and its surface is compatible with cell culture. At the same time, compared with the traditional culture dish, PDMS chip can provide a more controllable microenvironment for cell culture[46⇓⇓⇓~50].
As shown in Figure 4 (a), our group constructed a dual concentration gradient structure using PDMS[16]. The structure can simultaneously form nine groups of symmetrical concentration gradients and is used for studying the effect of the anticancer drug paclitaxel on tumor cells (MCF-7 and HepG2). The device reduces the environmental interference in the cell culture process, systematically and quantitatively studies the formation of concentration gradient based on the unique double-helix mixer, realizes the simultaneous evaluation of the effect of paclitaxel on two tumor cell lines, and carries out real-time observation and analysis on the activity of two tumor cells after 24 hours of action of different doses of drugs. The device can reduce the complexity of operation, thereby laying the foundation for establishing a low-cost laboratory chip platform in concentration gradient related biochemical research. In addition, as shown in Figure 4 (B), G Gómez-Sjoberg et al. established a fully automatic cell culture screening system based on PDMS chip, and quantitatively analyzed the effects of transient stimulation on the proliferation, osteogenic differentiation and movement of human primary mesenchymal stem cells[51]. The device enables the simultaneous creation of multiple media formulations in 96 separate culture chambers and keeps cells alive for weeks. Finally, the optimal culture conditions can be determined according to the cell inoculation density and the composition of the culture medium. In recent years, organ microarray construction and environmental pollution analysis based on PDMS concentration gradient system have become a hot research topic. As shown in fig. 4 (C), Zhang et al. describe a chemical gradient generator assisted microfluidic cell system for dynamic study of bronchial epithelial injury induced by representative environmental pollutants[52]. The device not only demonstrates the stability and reliability of operationally optimized microfluidic systems for precision and long-term chemical gradient production, but also performs microenvironmentally controlled microfluidic bronchial epithelial construction with high activity and structural integrity. In addition, the device can also be used to study the damage of bronchial epithelium stimulated by different concentrations of benzopyrene. According to the established system, different bronchial inflammatory and cytotoxic reactions were monitored and measured in time, and the inflammation and cytotoxicity induced by benzo (a) pyrene were successfully evaluated.
PDMS is easier to prepare and process than silicon or glass templates, and its cost is lower. At the same time, PDMS is widely used in biological reaction research such as cell culture, cell screening and biochemical analysis because of its gas permeability and surface compatibility with cell culture. However, the material also has some limitations, such as the incompatibility with organic solvents due to the permeability of gases, and the application of PDMS devices in aqueous solutions is also limited to a certain extent.

4 Application of Concentration Gradient Microfluidic Chip

4.1 Single Cell Analysis

Strategies for single cell isolation and manipulation play an extremely important role in disease diagnosis, drug delivery, and cancer biology[53]. Usually, homogeneous cells have a wide range of exogenous responses in response to specific drugs, which can not be observed by traditional drug screening methods, and the heterogeneity of individual cells may be crucial to the survival of cells. Methods that use population average responses often mask differences in individual cells. To fully understand cell-to-cell variability, a comprehensive analysis of individual cells, from their live state to cell lysates, is essential[54,55]. However, the traditional single cell analysis technology has the characteristics of high processing complexity and time-consuming, and requires expensive equipment, which greatly limits its application in the study of cell heterogeneity[56]. Techniques to rapidly and reliably assess drug resistance at the single-cell level and to monitor the extent of resistance from the intrinsic microenvironment of a single cell are still lacking[57]. High-sensitivity detection of multiple components and high-throughput analysis of large numbers of individual cells remain key challenges in achieving this goal. In this context, microfluidics and lab-on-a-chip have shown great advantages, and the development of microfabrication of microfluidic systems and increasingly sensitive molecular amplification tools has made it possible to miniaturize single-cell analysis platforms, which has become the most promising new technology to solve these problems.
Chung et al. proposed a microfluidic device as shown in fig. 5 (a) containing 64 single cell culture arrays to study cellular heterogeneity during multiple cell divisions, which captures individual cells into a microwell array, incubates the cells to allow clonal expansion, and subsequently drug therapy response[58]. For ease of operation, the prototype is designed to operate independently using a hydrodynamic cell capture method that is capable of capturing more than 80% of single cells into a single micropore using a natural flow mechanism driven by fluid resistance differences and gravity, and does not require an external energy source/pump for cell manipulation. In addition, to explore the potential of the device for assessing heterogeneous cell populations in cancer, single PC3 prostate cancer cells were captured and cultured in microwells, three different subphenotypes were identified in cancer models, chemotherapy was performed, and their clonal drug responsiveness was correlated with the cell phenotype. Liang et al. Designed a microfluidic chip, which provides a new tool for drug resistance monitoring and control at the single cell level[59]. As shown in fig. 5 (B), the chip integrates 549 cell traps and a microfluidic mixer to explore the effects of different concentrations of nitrogen on Molm-13 cells at the single-cell level. The design has high sensitivity, and has important clinical application prospect in single cell quality evaluation and drug analysis. As shown in fig. 5 (C), Song et al. developed a single-cell microfluidic device capable of static and dynamic concentration gradient distribution[60]. It is capable of generating dynamic temporal stimuli in a single device, observing the extracellular signal-regulated kinase (ERK) activity of epidermal growth factor (EGF) under pulse and ramp stimulation, and quantifying the activation of ERK at very low EGF concentrations, which is difficult to observe with conventional techniques. In addition, the combination of the microfluidic device and the living cell imaging technology can not only provide multifunctional stimulation for at least 12 hours, but also facilitate the long-term real-time observation of cell response at the single cell level, which is suitable for the study of single cell dynamics. Furthermore, Fernandes et al. Developed a flexible single-cell microfluidic device, which provides a powerful method for studying single-cell resolution and high throughput using transgenic yeast cells[61]. As shown in fig. 5 (d), the device contained a chemical gradient generator and nine chambers containing cell traps to study the protein alpha-synuclein production and aggregation in Saccharomyces cerevisiae. The study analyzed the combined effects of environmental factors and the expression levels of the protein alpha-synuclein by capturing individual cells. Pei et al. Proposed a microfluidic chip based on drug sensitivity (DS-chip)[62]. As shown in Figure 5 (e), the chip consists of a drug gradient generator and a parallel cell trap, enabling continuous single circulating tumor cell (CTC) capture, drug gradient distribution, drug stimulation, fluorescent probe labeling, and three-color fluorescence imaging. Using this device, the optimal drug response dose of single CTC was determined in 5 cancer patients. The device allows non-invasive and real-time measurement of drug sensitivity of tumor cells in patients during treatment, and can effectively guide drug selection and efficacy evaluation of personalized medication.
Microfluidic chips provide a convenient platform for single cell analysis, which is very beneficial for the study of single cell heterogeneity and its response to specific drugs. However, when monitoring and analyzing the biological behavior of a single cell, it is often necessary to combine other analysis equipment and display equipment in order to establish a more comprehensive analysis method and standardized processing process, but there are still some difficulties in integration.

4.2 Combination drug screening

In recent years, with the widespread use of various antibiotics and anti-cancer drugs, some cells and bacteria have developed a certain degree of drug resistance, and even "super bacteria" resistant to antibiotics have emerged.Therefore, more and more attention has been paid to the study of the inhibitory effect of the combination therapy of two or more antibiotics and anticancer drugs on multi-drug resistant pathogens and cancer cells[63⇓⇓⇓~67]. However, large-scale screening requires expensive and complex liquid handling systems for automation, thus reducing accessibility for clinicians or biologists and limiting the potential of sequential drug combinations for clinical applications and academic research. The use of microfluidic chips for drug screening allows space and time to control multiple microenvironments, reduces time-consuming operations in high-throughput large-scale screening, and widens the scope of application of large-scale screening of precision drugs. In addition, the microfluidic chip can also be used for comprehensive analysis of parallel experiments, control and multi-dose therapy in combination drug screening[68,69].
As shown in fig. 6 (a), combination drug therapy can produce a more powerful inhibitory effect than the use of a single drug, which acts through a specific pathway, while multiple drugs act through multiple pathways,In addition, single drug therapy requires a large dose, which is toxic to normal cells, while the synergistic effect of different drug combinations can reduce the dose of single drug, thereby reducing toxicity[70]. Therefore, it is very important to determine the appropriate concentration and type of drugs for the development of effective combination therapy. Microfluidic chips can achieve this function by using their special fluid characteristics, and microfluidic drug screening devices have attracted wide attention. As shown in fig. 6 (B), Kim et al. proposed a programmable, fully automated microfluidic cell array that integrates on-chip cell culture, drug concentration generation, and pair-wise combination.Cells can be cultured and exposed to different drugs in pair-wise concentration combinations in 64 separate cell culture chambers sequentially or simultaneously without continuous perfusion of drug-containing media[71]. The utility of this platform was demonstrated by sensitizing PC-3 human bone metastatic prostate cancer cells with two clinically relevant sensitizing agents, doxorubicin or mitoxantrone, or by simultaneously exposing the cells to a paired combination of a sensitizer and a tumor necrosis factor-related apoptosis-inducing ligand. At the same time, the ability of the device to perform continuous and simultaneous treatment also contributes to the exploration of different doses in toxicology and biology. Although the use of combination therapies usually produces a more powerful effect than the use of a single therapy (synergistic effect), some combinations may interfere, resulting in suppression of the effect (antagonism). As shown in fig. 6 (C), Kim et al. prepared a microfluidic-based phenotypic screening chip to study the combined effects of antibiotics by automatically generating two orthogonal concentration gradients on a bacteria-captured agarose gel.The simultaneous establishment of validation concentration combinations and various types of analysis for different antibiotics promotes the stability and effectiveness of drug testing on a chip[72]. In addition, our research group has developed a microfluidic chip device with wide flow range control, as shown in Figure 6 (d), which can quickly construct three concentration gradients of multiple solutes based on Dean flow within the flow range of 0.1~200μL·min-1, and conduct systematic and quantitative research and analysis through chemical gradient dynamics.Three stable, accurate and controllable drug gradients were established, and the individual effects and interactions of anticancer drugs (doxorubicin and cisplatin) on tumor cell lines (MCF-7 and HepG2) under the same conditions were successfully evaluated, which promoted the development of specific drug screening tools for tumor cells[15].
Combinatorial drug screening chip can realize the multiple concentration gradient screening of single drug and multiple drug combinations, and can also explore the synergistic and antagonistic effects in drug combinations. However, with the discovery of a large number of new targets and lead compounds,The efficiency of drug research and screening needs to be further improved. At the same time, there is still a gap between microfluidic drug screening models based on cells and molecules and in vivo models, so they can not fully reflect the pharmacological information of drugs.

4.3 3D cell culture

One of the drawbacks of current drug development techniques is the use of monolayer cell cultures, where the therapeutic outcome of a drug at a later stage (i.e., the clinical phase of drug development) is influenced by differences between 2D cell culture models in vitro and 3D cell structures in vivo. 3D cell culture devices constructed using microfluidic chips can simulate cell-cell and cell-matrix interactions and are physiologically more suitable for predicting drug responses to complex heterologous cancers.It also has significant potential in drug toxicity and efficacy analysis methods. At the same time, long-term spheroid culture and observation can be carried out by using microfluidic chip equipment, which makes multi-conditioned stimulation and in-situ analysis possible. In addition, microfluidic-based 3D cell culture combined with quantitative analysis can greatly improve the ability to simulate and understand the intracellular system[73].
As shown in Figure 7 (a), in 2D cell culture, cells are confined to the surface of a single material, mostly plastic (usually polystyrene), which has surface properties such as roughness, stiffness and limited binding sites, allowing cells to proliferate and migrate without restriction, which is not an accurate simulation of many cell in vivo conditions. The 3D cell culture platform allows cells to grow in all directions, unlike 2D cell culture, which restricts cells to a plane, 3D culture allows cells to adopt a preferred orientation to optimize cell polarity.Cell polarity can be changed in response to stimuli, signaling molecules and chemical gradients in the cell are not hindered, and the whole cell can be accessed, which is closer to the real environment in vivo[74]. Toh et al. Developed a microfluidic 3D hepatocyte chip device, as shown in fig. 7 (B), which contains multiple microfluidic channels, each of which has a 3D microenvironment to maintain the synthesis and metabolism of hepatocytes.And multiple channels allow multiple drug doses to be administered simultaneously to functional primary hepatocytes, while the addition of a concentration gradient generator allows dose-dependent drug responses in vitro to predict hepatotoxicity in vivo[75]. At the same time, the construction of multi-functional cell culture platform which integrates cell culture, microenvironment regulation and biochemical analysis has gradually become a research hotspot. As shown in fig. 7 (C), Sart et al. constructed a microfluidic platform for high-density 3D culture, controlled stimulation, and observation in a single chip[76]. The device extends the capabilities of droplet microfluidics for long-term culture of adherent cells. Multiscale cell analysis was performed using an array of 500 spheres per chip, in addition to extracting individual spheres for further analysis or culture, which will facilitate quantitative studies of 3D cultures under dynamic conditions and have implications for on-chip 3D cell culture, organ-on-chip, and cancer microenvironment studies.
The microfluidic chip is used to prepare 3D cells, which effectively simulates the growth characteristics of cells in vivo, and the research and analysis of cells in vitro is no longer limited to planar culture operation.It has shown great potential in physiological analysis and prediction of drug response. However, 3D cell culture is more complex, requires higher culture conditions, and has certain requirements for operators. In addition, there are still challenges in the effective recovery of 3D cultures and quantitative analysis.

4.4 Cell microenvironment simulation

Spatiotemporal regulation of the cell microenvironment plays an important role in monitoring cellular responses and exploring underlying physiological mechanisms.Up to now, the manipulation of the extracellular chemical microenvironment in vitro is usually accomplished by traditional (Petri dish or flask-based) cell culture systems, which lack precision and controllability[77⇓~79]. Microfluidic multi-organ chips simulate the microenvironment of tissue culture and realize the interconnection of organ equivalents, making this technology a powerful tool for preclinical drug screening[80].
As shown in fig. 8 (a), from replicating complex vasculature structures, co-culturing various cells, to mimicking cell-cell interactions, and simulating tumor exposure to shear stress, pressure, or biophysical and biochemical gradients, microfluidic tumor models have the unique advantage of recreating the complex microenvironment of a tumor in a relatively simple and representative manner[81]. In addition, the microfluidic tumor model can also be used for real-time monitoring, which provides a new platform for disease monitoring and drug screening[82]. As shown in Fig. 8 (B), Chen et al. proposed a new microwell array microfluidic system, which aims to achieve high-throughput culture of multicellular tumor spheres.The system constructs a bionic in vitro tumor microtissue model for drug screening research, and the columnar micropore array and the straight microchannel in the device can effectively transport nutrition and provide conditions for anti-cancer drug screening experiments[83]. In addition, due to the difficulty of cancer treatment and the heterogeneity of cells, it has attracted much attention to evaluate the response of different cancer cell lines to different drug combinations based on 3D cell microenvironment. Zhang et al. designed a scalable drug screening device based on 3D tumor model as shown in Figure 8 (C)[84]. The Christmas tree network in the device can generate a continuous concentration gradient between different drug pairs, and a tumor sphere is constructed to simulate the tumor microenvironment.The device has the advantages of low sample consumption and easy operation. In addition, the formation of concentration gradients and the cultivation of tumor spheres are of great significance for cell culture, precision drug action and high-throughput drug screening.
The microfluidic model summarizes a variety of physiologically relevant in vivo conditions, incorporates various characteristics of human tissues into the system, and constructs a more comprehensive and complete cell microenvironment.Current microfluidic models have made great strides in combining vasculature, cell type co-culture, and modeling the importance of elevated interstitial fluid pressure and shear stress. However, due to the small size of cells and the rapid diffusion of chemical molecules, as well as the structure of blood vessels, the artificial microenvironment in microfluidic chips is still a long way from the real environment of the human body in terms of the function of cell lines and simulation elements used in co-culture models.

5 Conclusion and prospect

In This paper, the characteristics of concentration gradient microfluidic chip are described, and the advantages and disadvantages of concentration gradient paper chip, concentration gradient hydrogel chip, concentration gradient droplet chip and concentration gradient PDMS chip are discussed.The applications of concentration gradient microfluidic chips in single cell analysis, combinatorial drug screening, 3D cell culture and cell microenvironment simulation are also described. Concentration-gradient microfluidic chips are time-saving, efficient, flexible materials and easy to control the structure, which provide a new technical method for drug screening. However, in the future development, there are still some problems to be solved, mainly including: (1) The system operation is complex, and there are certain requirements for technicians. (2) Although there are many choices of materials for microfluidic chips, there are still some limitations in their fabrication processes. (3) It is still difficult to integrate with other analysis equipment and display equipment.
As one of the rapidly developing high-tech and interdisciplinary research frontiers, the emergence of microfluidic chip analysis system makes it possible for experimental research to be miniaturized, integrated, automated and portable. Microfluidic chip analysis technology, which is used in biochemical analysis, cell culture, food detection and drug screening, has shown great potential and application value, and is gradually becoming a hot spot of attention and research. Microfluidic concentration gradient systems are also developing. Compared with traditional gradient generation methods, they have the advantages of saving time, reducing experimental costs, flexible control of concentration distribution and controllable analytical conditions. In addition, the microfluidic concentration gradient generator can also provide a microenvironment for the reaction of chemical signals, so the microfluidic concentration gradient generator has a wide range of applications in multiple drug screening, organ chips and biological research[85,86][87,88]. With the further improvement of precision medicine, the concentration gradient microfluidic chip will continue to provide an effective platform for drug screening and disease model development in future research, and further expand the availability of these systems, which will not only provide convenience in experimental applications, but also play an important role in practical engineering.

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Funding

National Natural Science Foundation of China(31700749)

Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi(2019L0362)

Science and Technology Innovation Funds of Shanxi Agricultural University(2016YJ02)

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