A biosensor method based on surfactant-mediated surface droplet evaporation for the detection of Aflatoxin B1.

Aflatoxin B1 (AFB1) is a common mycotoxin that is of significant global concern due to its impact on food safety. Herein, we innovatively develop a sensing platform to detect AFB1 based on evaporation of surfactant solutions on the hydrophobic surface, resulting in dried patterns with varied sizes. The surfactant CTAB solution produces a relatively large dried pattern due to the surface wetting. However, the reduction in the dried pattern size is found when the mixture of CTAB and AFB1 aptamer is tested, because the formation of CTAB/aptamer complex. Moreover, the dried pattern size of the mixture of CTAB, aptamer, and AFB1 increases due to the specific binding of AFB1 to its aptamer. Using this innovative strategy, the AFB1 detection can be fulfilled with a detection limit of 0.77 pg/mL. As a simple, convenient, inexpensive, and label-free method, the surfactant-mediated surface droplet evaporation-based biosensor is very promising for various potential applications.

Surfactant-mediated colorimetric assay assisted with in-situ rolling circle amplification on magnetic beads.

Gold nanoparticles (AuNPs) with localized surface plasmon resonance effect have been widely used for colorimetric detection based on the interparticle plasmon coupling during AuNPs aggregation. However, it is still challenging to develop portable and quantitative methods with good sensitivity and excellent selectivity. In this study, a smartphone-based colorimetric assay is developed on the principle of surfactant-mediated AuNPs aggregation assisted with rolling circle amplification (RCA) on magnetic beads (MBs). The detection of adenosine is demonstrated as an example. The cetyl trimethyl ammonium bromide (CTAB) causes the negatively charged AuNPs to aggregate, which results in the color change from red to blue. When adenosine is in solution, the RCA process is triggered on the MBs because of specific adenosine-aptamer recognition, resulting in prolongation of single-stranded nucleic acid (ssDNA). The solution color remains red due to the electrostatic interaction between CTAB and ssDNA. Using this method, the limit of detection (LOD) for adenosine can be as low as 16 pM. Besides, it also works well in human serum. In addition, a portable device integrated with in-situ RGB analysis software is developed for the detection with a smartphone. This study offers a new strategy to improve the sensitivity and selectivity for the AuNPs-based colorimetric assay, taking advantages of specific aptamer recognition, in-situ RCA on MBs, magnetic separation, and smartphone-based portable device.

Advances of microfluidic lung chips for assessing atmospheric pollutants exposure.

Atmospheric pollutants, including particulate matters, nanoparticles, bioaerosols, and some chemicals, have posed serious threats to the environment and the human's health. The lungs are the responsible organs for providing the interface betweenthecirculatory system and the external environment, where pollutant particles can deposit or penetrate into bloodstream circulation. Conventional studies to decipher the mechanismunderlying air pollution and human health are quite limited, due to the lack of reliable models that can reproduce in vivo features of lung tissues after pollutants exposure. In the past decade, advanced near-to-native lung chips, combining cell biology with bioengineered technology, present a new strategy for atmospheric pollutants assessment and narrow the gap between 2D cell culture and in vivo animal models. In this review, the key features of artificial lung chips and the cutting-edge technologies of the lung chip manufacture are introduced. The recent progresses of lung chip technologies for atmospheric pollutants exposure assessment are summarized and highlighted. We further discuss the current challenges and the future opportunities of the development of advanced lung chips and their potential utilities in atmospheric pollutants associated toxicity testing and drug screening.

Paper-Based Distance Sensor for the Detection of Lipase via a Phase Separation-Induced Viscosity Change.

Human pancreatic lipase is a symbolic biomarker for the diagnosis of acute pancreatitis, which has profound significance for clinical detection and disease treatment. Herein, we first demonstrate a paper-based lipase sensor via a phase separation-induced viscosity change. Lipase catalyzes triolein to produce oleic acid and glycerol. Adding an excess of Ca2+ produces calcium oleate. The remaining Ca2+ binds with sodium alginate, triggering hydrogelation with an "egg-box" structure. The viscosity change of the aqueous solution induced by the phase separation process can be quantified by measuring the solution flow distance on a pH test paper. The paper-based lipase sensor has high sensitivity with a detection limit of 0.052 U/mL and also shows excellent specificity. Additionally, it is also utilized for quantitative lipase analysis in human serum samples to exhibit its potency in acute pancreatitis detection. This method overcomes the drawbacks of low sensitivity, slow response, and poor reproducibility caused by the nonuniform distribution of the highly viscous hydrogel on the sensing interface in existing approaches. In conclusion, thanks to the prominent characteristics of high portability, low cost, and easy operation, it is prospective for simple quantitative detection of lipase and has great potential for commercialization.

Surface-anchored liquid crystal droplets for the semi-quantitative detection of Aflatoxin B1 in food samples.

Aflatoxin B1 (AFB1) is a common food mycotoxin that can cause various diseases. Therefore, reliable detection methods are required to ensure food safety against mycotoxins. In this study, we design a liquid-crystal (LC)-based assay for rapid detection of AFB1 in food samples. The surface-anchored LC droplets on glass (5CBSADrop) are obtained via a solvent evaporation method. The 5CBSADrop displays a four-leaf clover appearance that corresponds to an escape-radial configuration in a mixture of CTAB and AFB1 aptamer. Interestingly, they adopt a radial configuration in the mixture of CTAB, AFB1, and its aptamer. Using this approach, AFB1 can be detected using only 1 µL of the aqueous solution with a minimum detection concentration of 10 pg/mL. This LC-based sensing platform provides simple operation, remarkable sensitivity, high selectivity, low cost, and excellent portability without the use of any bulky instrument, which is very promising in rapid on-field detection of mycotoxins.

Liquid crystal-based sensitive and selective detection of uric acid and uricase in body fluids.

The abnormal levels of uric acid (UA) in body fluids are associated with gout, type (II) diabetes, leukemia, Lesch-Nyhan syndrome, uremia, kidney damage, and cardiovascular diseases. Also, the presence of uricase (UOx) symbolizes genetic disorders and corresponding complications. Therefore, the detection of UA and UOx in the body fluids is significant for clinical diagnosis. 4-Cyano-4'-pentylbiphenyl (5CB, a nematic liquid crystal (LC)) was doped with octadecyl trimethylammonium bromide (OTAB, a cationic surfactant), which formed a self-assembled monolayer at the aqueous/5CB interface. The UOx-catalyzed oxidation of UA yielded H2O2, releasing the single-strand deoxyribonucleic acid (ssDNA) from the nanoceria/ssDNA complex. The interaction of the released ssDNA with OTAB disrupted the monolayer at the aqueous/5CB interface, which resulted in a dark to bright change when observed through a polarized optical microscope. The LC-based sensor allowed the detection of UA with a linear range of 0.01-10 µM and a limit of detection (LOD) of 0.001 µM. The UA detection was also performed in human urine samples and the results were comparable to that of a standard commercial colorimetric method. Similarly, the detection of UOx was performed, with a noted linear range of 20-140 µg/mL. The LOD was as low as 0.34 µg/mL. The detection of UOx was also demonstrated in human serum samples with excellent performance. This method provides a robust sensing platform for the detection of UA and UOx and has potential for applications in clinical analysis.

A portable digital optical kanamycin sensor developed by surface-anchored liquid crystal droplets.

There is an increase in demand to develop simple, convenient, and low-cost approaches for rapid and label-free detection of antibiotics. Herein, we propose a new principle for the detection of kanamycin using the surface-anchored liquid crystal (LC) droplets. The optical images of the LC droplets uniformly change from four-clover, uniformly dark, and dark cross appearance gradually with the increase of surfactant concentration. The detection of kanamycin is fulfilled with the aid of a cationic surfactant cetyltrimethylammonium bromide (CTAB) and a kanamycin aptamer. The LC droplets show uniformly dark appearance and four-clover appearance in the presence of the aqueous solutions of CTAB and CTAB/aptamer complex, respectively. However, the specific binding of kanamycin to its aptamer can release the CTAB, which induces the uniformly dark appearance of the LC droplets. A portable device is built to measure the optical luminance of the LC droplets. This system can detect kanamycin with a concentration below 0.1 ng/mL (~0.17 nM) and also allows the detection of kanamycin in real samples such as milk and honey. Therefore, it is very promising in the development of new types of LC-based sensors by the surface-anchored LC droplets assisted with a portable optical device.

HiPSC-derived multi-organoids-on-chip system for safety assessment of antidepressant drugs.

The poor predictive power of existing preclinical models has spurred efforts to develop human-relevant models for accurate assessment of drug safety. In this work, we developed a multi-organoids-on-a-chip system derived from human induced pluripotent stem cells (hiPSCs), which allows for the assessment of the cardiac safety of an antidepressant drug, following liver metabolism in vitro. This liver-heart organoids-on-chip device contains compartmentalized chambers separated by a porous membrane, which permits the co-culture of 3D human liver organoids in the upper multi-well chamber and cardiac organoids in the bottom micropillar array simultaneously. The co-cultured liver and heart organoids on chip maintained good viability and human organ-specific functions respectively, including the synthesis of albumin and urea of liver organoids, and the beating function of cardiac organoids. In particular, the liver organoids displayed proper metabolic capabilities with high expression of CYP450 enzyme genes. Clomipramine, a widely used antidepressant drug, can be metabolized into an active metabolite (desmethylclomipramine) through the hepatic CYP450 enzymes of liver organoids on chip identified by mass spectrometry. After exposure to 1 µM clomipramine in the liver chamber for 24 h and 48 h, the co-cultured heart organoids in the bottom layer showed significantly reduced cell viability, impaired functions of cardiac beating and calcium flux, indicating the hepatic metabolism-dependent cardiotoxicity induced by clomipramine. By combining stem cell biology and microengineered technology, this proposed hiPSC-derived multi-organoids-on-a-chip system can reflect human organ-specific functions, as well as the complex process of drug metabolism and responses at the multi-organ level. It may provide a novel platform for the assessment of drug effectiveness and safety in vitro.

Amnion-on-a-chip: modeling human amniotic development in mid-gestation from pluripotent stem cells.

The amnion serves to create a protective environment for a growing fetus, and the study of amniotic development will greatly facilitate our understanding of normal and abnormal pregnancies. However, this remains a poorly studied field due to the lack of ideal human models. Herein, we present an integrative strategy to generate amnion-like cavity tissue from human pluripotent stem cells (hPSCs) in an amnion-on-a-chip device through combining a bioengineering approach and developmental biology principles. hPSCs could self-organize into an amnion epithelial cavity in a perfusable 3D culture microchip, resembling human amniotic development in mid-gestation. These cavities exhibited the critical features of amnion tissue based on morphological characteristics, marker expression, and transcriptome analysis. RNA-seq revealed that a set of amnion-specific genes were highly expressed in the obtained cavities, suggesting that the amnion epithelium was derived from hPSCs. Moreover, the amnion-specific mid-gestation marker KRT24 was highly expressed at the mRNA and protein levels, verifying the high maturation of amnion tissues after long-term 3D culturing and differentiation for up to 20 days. These new findings demonstrate the potential of this new amnion-on-a-chip model for investigating essential biological events in human amnions in normal and diseased states via integrating microengineering technology and stem cell biology.

Neurodevelopmental impairment induced by prenatal valproic acid exposure shown with the human cortical organoid-on-a-chip model.

Prenatal exposure to environmental insults can increase the risk of developing neurodevelopmental disorders. Administration of the antiepileptic drug valproic acid (VPA) during pregnancy is tightly associated with a high risk of neurological disorders in offspring. However, the lack of an ideal human model hinders our comprehensive understanding of the impact of VPA exposure on fetal brain development, especially in early gestation. Herein, we present the first report indicating the effects of VPA on brain development at early stages using engineered cortical organoids from human induced pluripotent stem cells (hiPSCs). Cortical organoids were generated on micropillar arrays in a controlled manner, recapitulating the critical features of human brain development during early gestation. With VPA exposure, cortical organoids exhibited neurodevelopmental dysfunction characterized by increased neuron progenitors, inhibited neuronal differentiation and altered forebrain regionalization. Transcriptome analysis showed new markedly altered genes (e.g., KLHL1, LHX9, and MGARP) and a large number of differential expression genes (DEGs), some of which are related to autism. In particular, comparison of transcriptome data via GSEA and correlation analysis revealed the high similarity between VPA-exposed organoids with the postmortem ASD brain and autism patient-derived organoids, implying the high risk of autism with prenatal VPA exposure, even in early gestation. These new findings facilitate a better understanding of the cellular and molecular mechanisms underlying postnatal brain disorders (such as autism) with prenatal VPA exposure. This established cortical organoid-on-a-chip platform is valuable for probing neurodevelopmental disorders under environmental exposure and can be extended to applications in the study of diseases and drug testing.

Microengineered hiPSC-Derived 3D Amnion Tissue Model to Probe Amniotic Inflammatory Responses under Bacterial Exposure.

Intra-amniotic infection is a common cause of preterm birth that can lead to adverse neonatal outcomes. Despite the basic and clinical significance, the study in normal and diseased human amnion is highly challenging due to the limited use of human primary tissues and the distinct divergence between animal models and human. Here, we established a microengineered hiPSC-derived amnion tissue model on a chip to investigate the inflammatory responses of amnion tissues to bacterial exposure. The microdevice consisted of two parallel channels with a middle matrix channel, creating a permissive microenvironment for amnion differentiation. Dissociated hiPSCs efficiently self-organized into cell cavity and finally differentiated into a polarized squamous amniotic epithelium on the chip under perfused 3D culture. When exposed to E. coli, amnion tissue exhibited significant functional impairments compared to the control, including induced cell apoptosis, disrupted cell junction integrity, and increased inflammatory factor secretion, recapitulating a series of characteristic clinical signs of intra-amniotic infection at an early stage. Together, this amnion-on-a-chip model provides a promising platform to investigate intrauterine inflammation in early gestation, indicating its potential applications in human embryology and reproductive medicine.

A 3D human placenta-on-a-chip model to probe nanoparticle exposure at the placental barrier.

With the commercialization of nanomaterials, environmental exposure to nanoparticles (NPs) has raised great concerns due to the long-term effects to human body, particularly to pregnant women. Previous studies found that NPs had an adverse impact on placenta in mice, but care must be taken when extrapolating the results to human pregnancy in consideration of the great difference between species. Here, we proposed a microengineered 3D placental barrier-on-a-chip microdevice and further explored complicated placental responses to NPs exposure in vitro. The microdevice recreated near-physiological 3D microenvironment and dynamic conditions in fetal maternal circulation combined with the extracellular matrix and flow. With the exposure to titanium dioxide nanoparticles (TiO2-NPs), a common nanomaterial, a series of placental responses were investigated, including oxidative stress, cell apoptosis, barrier permeability, and maternal immune cell behavior. By contrast to oxidative stress and cell apoptosis, placental barrier integrity and maternal immune cells were greatly influenced even with low concentrated NPs, suggesting the potential damages triggered by NPs in our daily life. Collectively, this in vitro experimental model of human placenta provides a simple platform to study environmental exposure to NPs, and might be potential for a wide range of applications in biological study, disease treatment and drug assessment.

Placental Barrier-on-a-Chip: Modeling Placental Inflammatory Responses to Bacterial Infection.

Placental inflammation, as a recognized cause of preterm birth and neonatal mortality, displays extensive placental involvement or damage with the presence of organisms. The inflammatory processes are complicated and tightly associated with increased inflammatory cytokine levels and innate immune activation. However, the deep study of the underlying mechanisms was limited by conventional cell and animal models because of great variations in the architecture and function of placenta. Here, we established a microengineered model of human placental barrier on the chip and investigated the associated inflammatory responses to bacterial infection. The multilayered design of the microdevice mimicked the microscopic structure in the fetal-maternal interfaces of human placenta, and the flow resembled the dynamic environment in the mother's body. Escherichia coli (E. coli), one of the predominant organisms found in fetal organs, were applied to the maternal side, modeling acute placental inflammation. The data demonstrated the complex responses including the increased secretion of inflammatory cytokines by trophoblasts and the adhesion of maternal macrophages following bacterial infection. Particularly, transplacental communication was observed between two placental cells, and implied the potential role of trophoblast in fetal inflammatory response syndrome in clinic. These complex responses are of potential significance to placental dysfunctions, even abnormal fetal development and preterm birth. Collectively, placental barrier-on-a-chip microdevice presents a simple platform to explore the complicated inflammatory responses in human placenta, and might help our understanding of the mechanisms underlying reproductive diseases.

Engineering Brain Organoids to Probe Impaired Neurogenesis Induced by Cadmium.

Brain organoids derived from human induced pluripotent stem cells (hiPSCs) are three-dimensional in vitro models with near-physiological cellular composition and structural organization, which is representative of the developing human brain. They provide an ideal experimental system for the investigation of brain development and diseases. Prenatal exposure to the heavy metal cadmium (Cd) poses a serious health threat, particularly to the developing brain due to a long biological half-life of Cd in vivo. Although it is known that prolonged exposure to Cd will cause toxic effects because of its low rate of excretion from the body, the underlying mechanisms of Cd neurotoxicity remain unclear. Herein, we proposed a simple approach to engineer brain organoids on an array chip with octagon-shaped micropillars and explored neural dysfunctions of brain organoids under Cd exposure. hiPSC-derived brain organoids with millimeter-size recapitulated spatial and temporal patterning events in the early developing brain, including gene expression programs and three-dimensional organization. With Cd exposure, brain organoids displayed induced cell apoptosis, skewed neural differentiation, and varied brain regionalization, indicating the presence of impaired neurogenesis in the human fetal brain. This work provides a simple manner to generate brain organoids efficiently and a powerful platform for the investigation of abnormal neurogenesis induced by many different toxic factors in vitro.

Stimulation of chondrocytes and chondroinduced mesenchymal stem cells by osteoinduced mesenchymal stem cells under a fluid flow stimulus on an integrated microfluidic device.

The aim of the present study was to investigate the stimulation of osteoinduced mesenchymal stem cells (MSCs) into chondrogenically predifferentiated MSCs and chondrocytes in a mechanical environment. A novel two­layer microfluidic chip was used to mimic the interstitial flow in the superficial zones of articular cartilage. The morphology, proliferation rate and the expression of collagen I, collagen II and aggrecan of chondrocytes and chondro­MSCs were investigated. The results revealed that the cells in the bottom layer were influenced by the top layer's osteoinduced MSCs and the bottom layer's shear flow. The expression of collagen I, which may signify the effect of the shear stress on the dedifferentiation change, was weakened by the stimulation of osteoinduced MSCs on the top layer. The expression of collagen II and aggrecan was increased in the fluidic environment by osteoinduced MSCs. These results indicate that osteoinduced MSCs have a significant effect on the phenotype of chondro­MSCs and chondrocytes in the fluidic microenvironment. The present study described a simple and promising way to rapidly evaluate cell responses to other cells in a fluidic environment, which may help to better promote the utilization of MSCs and chondrocytes in tissue engineering.

Probing impaired neurogenesis in human brain organoids exposed to alcohol.

The fetal brain is highly vulnerable to ethanol exposure, which can trigger various long-term neuronal disabilities and cognitive dysfunctions. However, a comprehensive understanding of fetal brain development under ethanol exposure is challenging due to the limitations of animal models. Here, we propose a human induced pluripotent stem cell (hiPSC)-based 3D brain organoid model, and explore the mechanisms underlying neural dysfunctions in prenatal alcohol exposure (PAE) in vitro. Brain organoids were examined to resemble brain organogenesis in vivo at early stages during gestation, with specific features of neuronal differentiation, brain regionalization, and cortical organization. With ethanol exposure, the brain organoids displayed attenuated neurite outgrowth and skewed neural maturation. Transcriptome analysis identified a series of new markedly altered genes and enriched pathways, such as GSX2, RSPO2, and the Hippo signaling pathway. These genes or pathways, to our knowledge, were reported to be involved in ethanol-induced impaired neurogenesis for the first time. Our new findings might facilitate better understanding of the various postnatal neural disorders observed in individuals with PAE.

A hollow fiber system for simple generation of human brain organoids.

3D organoids exhibit near-physiological morphogenesis and histology relying on the self-organization of human pluripotent stem cells (hPSCs), representing a new class of in vitro model for studying developmental biology and diseases. An engineered approach is highly desirable to generate sufficient organoids in a simple and efficient manner. Herein, we present a new strategy for the simple formation of massive human brain organoids from hiPSCs within a hollow fiber reactor system by combining fiber materials with the developmental biology principle. A thin and finely adjustable calcium alginate (CaA) core-shell fiber was constructed using a multilayer coaxial laminar flow microfluidic system. The meter-long hollow fibers enabled neural differentiation of hiPSCs and simple formation of abundant brain organoids in a 3D matrix. The generated brain organoids displayed essential features of human brain organogenesis, including polarized neuroepithelium, cell type heterogeneity and discrete brain regions, resembling the early brain development. This approach is simple and easy to operate, which allows for simplified formation of massive brain organoids, overcoming the tedious procedures in conventional methods. In particular, the facile and scalable characteristics of hollow fibers are compatible with real-time observation and monitoring, as well as flexible tissue manipulations for downstream biological analysis. It might also provide a new platform to advance stem cell-derived organoid models and their utility in biomedical applications.

In situ generation of human brain organoids on a micropillar array.

Brain organoids derived from human induced pluripotent stem cells can recapitulate the early stages of brain development, representing a powerful in vitro system for modeling brain development and diseases. However, the existing methods for brain organoid formation often require time-consuming procedures, including the initial formation of embryoid bodies (EBs) from hiPSCs, and subsequent neural induction and differentiation companied by multi-steps of cell transfer and encapsulation in a 3D matrix. Herein, we propose a simple strategy to enable in situ formation of massive brain organoids from hiPSCs on a micropillar array without tedious manual procedures. The optimized micropillar configurations allow for controlled EB formation, neural induction and differentiation, and generation of functional human brain organoids in 3D culture on a single device. The generated brain organoids were examined to imitate brain organogenesis in vivo at early stages of gestation with specific features of neuronal differentiation, brain regionalization, and cortical organization. By combining microfabrication techniques with stem cells and developmental biology principles, the proposed method can greatly simplify brain organoid formation protocols as compared to conventional methods, overcoming the potential limitations of cell contamination, lower throughput and variance of organoid morphology. It can also provide a useful platform for the engineering of stem cell organoids with improved functions and extending their applications in developmental biology, drug testing and disease modeling.

Bioinspired onion epithelium-like structure promotes the maturation of cardiomyocytes derived from human pluripotent stem cells.

Organized cardiomyocyte alignment is critical to maintain the mechanical properties of the heart. In this study, we present a new and simple strategy to fabricate a biomimetic microchip designed with an onion epithelium-like structure and investigate the guided behavior of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) on the substrate. The hiPSC-CMs were observed to be confined by the three dimensional surficial features morphologically, analogous to the in vivo microenvironment, and exhibited an organized anisotropic alignment on the onion epithelium-like structure with good beating function. The calcium imaging of hiPSC-CMs demonstrated a more mature Ca2+ spark pattern as well. Furthermore, the expression of sarcomere genes (TNNI3, MYH6 and MYH7), potassium channel genes (KCNE1 and KCNH2), and calcium channel genes (RYR2) was significantly up-regulated on the substrate with an onion epithelium-like structure instead of the surface without the structure, indicating a more matured status of cardiomyocytes induced by this structure. It appears that the biomimetic micropatterned structure, analogous to in vivo cellular organization, is an important factor that might promote the maturation of hiPSC-CMs, providing new biological insights to guide hiPSC-CM maturation by biophysical factors. The established approach may offer an effective in vitro model for investigating cardiomyocyte differentiation, maturation and tissue engineering applications.