Multiparameter Mechanical Phenotyping for Accurate Cell Identification Using High-Throughput Microfluidic Deformability Cytometry.

Mechanical phenotyping has been widely employed for single-cell analysis over recent years. However, most previous works on characterizing the cellular mechanical properties measured only a single parameter from one image. In this paper, the quasi-real-time multiparameter analysis of cell mechanical properties was realized using high-throughput adjustable deformability cytometry. We first extracted 12 deformability parameters from the cell contours. Then, the machine learning for cell identification was performed to preliminarily verify the rationality of multiparameter mechanical phenotyping. The experiments on characterizing cells after cytoskeletal modification verified that multiple parameters extracted from the cell contours contributed to an identification accuracy of over 80%. Through continuous frame analysis of the cell deformation process, we found that temporal variation and an average level of parameters were correlated with cell type. To achieve quasi-real-time and high-precision multiplex-type cell detection, we constructed a back propagation (BP) neural network model to complete the fast identification of four cell lines. The multiparameter detection method based on time series achieved cell detection with an accuracy of over 90%. To solve the challenges of cell rarity and data lacking for clinical samples, based on the developed BP neural network model, the transfer learning method was used for the identification of three different clinical samples, and finally, a high identification accuracy of approximately 95% was achieved.

Inertial Multi-Force Deformability Cytometry for High-Throughput, High-Accuracy, and High-Applicability Tumor Cell Mechanotyping.

Previous on-chip technologies for characterizing the cellular mechanical properties often suffer from a low throughput and limited sensitivity. Herein, an inertial multi-force deformability cytometry (IMFDC) is developed for high-throughput, high-accuracy, and high-applicability tumor cell mechanotyping. Three different deformations, including shear deformations and stretch deformations under different forces, are integrated with the IMFDC. The 3D inertial focusing of cells enables the cells to deform by an identical fluid flow, and 10 parameters, such as cell area, perimeter, deformability, roundness, and rectangle deformability, are obtained in three deformations. The IMFDC is able to evaluate the deformability of different cells that are sensitive to different forces on a single chip, demonstrating the high applicability of the IMFDC in analyzing different cell lines. In identifying cell types, the three deformations exhibit different mechanical responses to cells with different sizes and deformability. A discrimination accuracy of ≈93% for both MDA-MB-231 and MCF-10A cells and a throughput of ≈500 cells s-1 can be achieved using the multiple-parameters-based machine learning model. Finally, the mechanical properties of metastatic tumor cells in pleural and peritoneal effusions are characterized, enabling the practical application of the IMFDC in clinical cancer diagnosis.

Elasto-inertial particle focusing in sinusoidal microfluidic channels.

Dean flow existing in sinusoidal channels could enhance the throughput and efficiency for elasto-inertial particle focusing. However, the fundamental mechanisms of elasto-inertial focusing in sinusoidal channels are still unclear. This work employs four microfluidic devices with symmetric and asymmetric sinusoidal channels to explore the elasto-inertial focusing mechanisms over a wide range of flow rates. The effects of rheological property, flow rate, sinusoidal channel curvature, particle size, and asymmetric geometry on particle focusing performance are investigated. It is intriguing to find that the Dean flow makes a substantial contribution to the particle elasto-inertial focusing. The results illustrate that a better particle focusing performance and a faster focusing process are obtained in the sinusoidal channel with a small curvature radius due to stronger Dean flow. In addition, the particle focusing performance is also related to particle diameter and rheological properties, the larger particles show a better focusing performance than smaller particles, and the smaller flow rate is required for particles to achieve stable focusing at the outlet in the higher concentration of polyvinylpyrrolidone solutions. Our work offers an increased knowledge of the mechanisms of elasto-inertial focusing in sinusoidal channels. Ultimately, these results provide supportive guidelines into the design and development of sinusoidal elasto-inertial microfluidic devices for high-performance focusing.

Particle focusing mechanisms in λ-DNA solution flowing in a straight microchannel.

Most biological fluids (such as blood, saliva, and lymph) in nature have certain viscoelasticity and are beginning to be used as the carrying fluids for viscoelastic microfluidics. However, the particle-focusing mechanisms in these new biological viscoelastic fluids are still unclear. In this work, the particle-focusing mechanisms in λ-DNA solutions were systematically explored. We first explored the particle focusing dynamics in a square cross-section under varied flow rates to uncover the effects of flow rate on particle focusing. Three focusing stages, from the classic five-position viscoelastic focusing to single-stream focusing and finally to multiplex-stream focusing, were clearly demonstrated. In addition, the particle focusing process along the channel length was demonstrated, and a first-fast-and-then-slow focusing process was clearly observed. Then, the effects of λ-DNA concentrations on particle focusing were explored and compared using the solutions with 0-25 ppm λ-DNA. Finally, we discussed the inferences of blockage ratio on particle focusing by changing the particle diameter and cross-sectional dimensions. Our work may provide a deeper understanding on the particle focusing mechanisms in biological viscoelastic fluids and lays a foundation for the subsequent particle counting and analysis and the development of low-cost portable flow cytometers.

Viscoelastic-Sorting Integrated Deformability Cytometer for High-Throughput Sorting and High-Precision Mechanical Phenotyping of Tumor Cells.

The counts and phenotypes of circulating tumor cells (CTCs) in whole blood are useful for disease monitoring and prognostic assessment of cancer. However, phenotyping CTCs in the blood is difficult due to the presence of a large number of background blood cells, especially some blood cells with features similar to those of tumor cells. Herein, we presented a viscoelastic-sorting integrated deformability cytometer (VSDC) for high-throughput label-free sorting and high-precision mechanical phenotyping of tumor cells. A sorting chip for removing large background blood cells and a detection chip for detecting multiple cellular mechanical properties were integrated into our VSDC. Our VSDC has a sorting efficiency and a purity of over 95% and over 81% for tumor cells, respectively. Furthermore, multiple mechanical parameters were used to distinguish tumor cells from white blood cells using machine learning. An accuracy of over 97% for identifying tumor cells was successfully achieved with the highest identification accuracy of 99.4% for MCF-7 cells. It is envisioned that our VSDC will open up new avenues for high-throughput and label-free single-cell analysis in various biomedical applications.

Next-generation liquid biopsy instruments: Challenges and opportunities.

Conventional cancer diagnosis needs to excise diseased tissue from the patient's body for biopsy, causing severe injury to patients. Liquid biopsy (LB), with the superior advantage of minimal invasiveness, has shown its ability to cancer diagnosis in real-time and has been developing promising diagnostic instruments. However, until today, the developed instrument still cannot be an alternative to tissue biopsy in the majority of research and clinical settings. In this paper, we first summarize the challenges and limitations suffered by the existing LB instrument. Then, the opportunities and future progression of the next-generation instrument are discussed in detail. In all, we hope that the future LB instrument can be eventually integrated into the clinical workflow and serve as a validated and reliable tool for cancer diagnosis.

Droplet microfluidics for CTC-based liquid biopsy: a review.

Circulating tumor cells (CTCs) are important biomarkers of liquid biopsy. The number and heterogeneity of CTCs play an important role in cancer diagnosis and personalized medicine. However, owing to the low-abundance biomarkers of CTCs, conventional assays are only able to detect CTCs at the population level. Therefore, there is a pressing need for a highly sensitive method to analyze CTCs at the single-cell level. As an important branch of microfluidics, droplet microfluidics is a high-throughput and sensitive single-cell analysis platform for the quantitative detection and heterogeneity analysis of CTCs. In this review, we focus on the quantitative detection and heterogeneity analysis of CTCs using droplet microfluidics. Technologies that enable droplet microfluidics, particularly high-throughput droplet generation and high-efficiency droplet manipulation, are first discussed. Then, recent advances in detecting and analyzing CTCs using droplet microfluidics from the different aspects of nucleic acids, proteins, and metabolites are introduced. The purpose of this review is to provide guidance for the continued study of droplet microfluidics for CTC-based liquid biopsy.

Boosting the performance of an iontophoretic biosensing system with a graphene aerogel and Prussian blue for highly sensitive and noninvasive glucose monitoring.

Diabetes and impaired glucose regulation (IGR) threaten the lives and health of numerous patients. Interstitial fluid (ISF) glucose, displaying an excellent correlation with blood glucose, is highly desired to address the limitations of invasive and minimally invasive glucose detection. Herein, we present a screen-printed iontophoretic biosensing system to extract ISF noninvasively and perform in situ instant glucose detection. A three-dimensional graphene aerogel combined with Prussian blue (GA@PB) was introduced as an electron mediator, providing suitable support for glucose oxidase (GOx) immobilization, highly boosting the detection sensitivity. Additionally, a self-made diffuse cell and an ex vivo model were developed to demonstrate the efficacy of ISF extraction based on reverse iontophoresis technology. Highly sensitive and accurate detection of ISF glucose could be achieved with an LOD of 0.26 mM over a 0-15 mM range. Finally, tests on healthy volunteers were conducted to further validate the feasibility of this as-proposed system. Combined with its well flexible and biocompatible features, it holds considerable prospects in the development of wireless wearable biosensors for continuous blood glucose monitoring.

Hemodynamic Mimic Shear Stress for Platelet Membrane Nanobubbles Preparation and Integrin αIIbß3 Conformation Regulation.

Platelet (PLT) membrane biomimetic nanomaterials have become promising theranostic platforms due to their good biocompatibility and effectiveness. However, in order to achieve precise regulation of cell membrane components, novel controllable construction approaches need to be developed. Inspired by the interaction mechanism among platelet production, activation, and dynamic biomechanical signals in blood circulation, here a platelet nanobubbles (PNBs) with reassembled platelet membrane with ideal echogenicity was fabricated using an adjustable pressure-induced shear stress method. The results demonstrate that the high shear stress during PNBs fabrication led to the enrichment of platelet membrane lipid rafts and proteins, as well as their reassembly on the gas-liquid interface. More importantly, the conformation of platelet integrin αIIbß3 was transformed into a shear stress-induced intermediate affinity state, which gives PNBs enhanced adhesion ability to the vascular endothelial injury. Taken together, these PNBs have great application potential in the specifically targeted ultrasound diagnosis of vascular endothelial injury.

Portable Battery-Driven Microfluidic Cell Separation Instrument with Multiple Operational Modes.

Current microfluidic cell separation instruments are commonly bulky, expensive, and inconvenient for use in resource-limited environments. Herein, we developed a low-cost, portable, battery-driven microfluidic cell separation instrument by integrating a microfluidic separator, a low-cost gas-driven flow system, and a flow resistance module. The plug-and-play cell separator enabled by inertial microfluidics was used to realize the label-free solid/solid or solid/liquid separation by switching the operational modes of cell separation and enrichment. To increase the instrument portability, an all-in-one flow resistance module integrated with three different polymer film flow resistance chips was designed to stabilize the gas flow generated by the miniaturized diaphragm pump for low-cost fluid injection under different operational modes. After successfully constructing our instrument, the performance under the separation mode was tested using the blood samples spiked with three different tumor cells. Then, the enrichment mode was performed to concentrate the target cells after separation and thus reduce the sample volume. Excellent separation and enrichment performances were achieved at a high throughput. Our portable microfluidic cell separation instrument offers the advantages of simple operation, small footprint, lightweight, and untethered battery drivenness and thus holds the potential for one-stop cell preparation in resource-limited environments.

A novel 3D Tesla valve micromixer for efficient mixing and chitosan nanoparticle production.

Current three-dimensional micromixers for continuous flow reactions and nanoparticle synthesis are complex in structure and difficult to fabricate. This paper investigates the design, fabrication, and characterization of a novel micromixer that uses a simple spatial Tesla valve design to achieve efficient mixing of multiple solutions. The flow characteristics and mixing efficiencies of our Tesla valve micromixer are investigated using a combination of numerical simulations and experiments. The results show that in a wide range of flow rates, viscoelastic solutions with different concentrations can be well mixed in our micromixer. Finally, experiments on the synthesis of chitosan nanoparticles are conducted to verify the practicability of our micromixer. Compared with nanoparticles prepared by conventional magnetic stirring, the size of nanoparticles prepared by micromixing is smaller and the distribution is more uniform. Therefore, our Tesla valve micromixer has significant advantages and implications for mixing chemical and biological reactions.

Glucose sensing on screen-printed electrochemical electrodes based on porous graphene aerogel @prussian blue.

As one of the three major chronic diseases, diabetes often causes many complications, which can affect various parts of the body and even threaten the life of the patients. At present, the situation of diabetes in the world is quite serious. Accurate detection of blood glucose is very important for the diagnosis, treatment and medication of diabetes as well as the self-management of diabetic patients. In this paper, an electrochemical glucose biosensor was developed based on screen-printed electrode (SPE) modified with composite material of graphene aerogel (GA) and Prussian blue (PB) (denoted as GA@PB), which was fabricated via chemical reduction using L-ascorbic acid as a reducing agent through a freeze-drying process. Glucose was specifically captured by glucose oxidase (GOx) which were immobilized into the GA@PB by chitosan. The structure and performance of the sensor were characterized by scanning electron microscopy (SEM), Raman spectroscopy measurements, Fourier transform infrared spectrometer (FTIR), cyclic voltammetry (CV) and amperometric detection. The sensor exhibited a linear range of 0.5-6.0 mmol·L-1 with limit of detection (LOD) of 0.15 mmol·L-1, indicating that the combination of graphene aerogel and Prussian blue possess well conductivity and catalytic performance.

Key Factors of Mechanical Strength and Toughness in Oriented Poly(l-lactic acid) Monofilaments for a Bioresorbable Self-Expanding Stent.

The investigation of the strength and toughness of poly(l-lactic acid) (PLLA) monofilaments is essential as the fundamental element of a biodegradable braided stent. However, the determining factor remains poorly addressed with respect to influencing the mechanical behavior of PLLA monofilaments. In this work, the electron beam (EB) with different radiation doses was utilized to sterilize PLLA monofilaments. Properties of the monofilaments, including the breaking strength, elongation at break, molecular weight, orientation, and microstructure of the fracture, were characterized. Results showed that a random chain scission of PLLA resulting from EB during this process could cause the decrease in molecular weight, which led to the decline in breaking strength. Meanwhile, the irradiated monofilaments were found to have almost the same elongation at break below a dose of 30 kGy and declined by 71.41% up to a dose of 48 kGy. It was also found that the ductile fracture connection of the monofilament translated to the brittle fracture by comparing the microstructure without and with sterilization. These phenomena could originate from the destruction of the long molecular chains connecting the crystal plates into shorter ones by radiation. PLLA monofilaments with 0, 30, and 48 kGy were used to braid carotid stents. Compared with a carotid Wallstent, the PLLA stent can better provide radial supporting to the carotid lesion. This study provides preliminary experimental references to evaluate and predict the mechanical performance of PLLA braided stents.

The elevation of serum uric acid depends on insulin resistance but not fasting plasma glucose in hyperuricaemia.

OBJECTIVES: The association between serum uric acid (SUA) and fasting plasma glucose (FPG) has not been fully outlined, in particular in hyperuricaemic population. This study aimed to address this issue, along with the exploration of the role of insulin resistance that was assessed by triglyceride-and-glucose (TyG) index. METHODS: A total of 16,297 participants without known diabetes from the SENSIBLE and SENSIBLE-Addition studies were included in the present analysis. Hyperuricaemia was defined as SUA ≥6 mg/dL. Generalised addictive model was applied to establish the relationship of SUA with FPG, and mediation analysis was performed to assess how insulin resistance affected the relationship. RESULTS: SUA showed an inverted U-shaped association with FPG, with the turning point of FPG at 6.1 mmol/L and 7.5 mmol/L in normouricaemic and hyperuricaemic participants, respectively. However, the significant relationship between SUA and FPG disappeared in hyperuricaemic participants (form B=3.3, 95% CI: 0.6-5.9, p=0.016 to B= -0.2, 95% CI: -3.1-2.7, p=0.894), and attenuated in normouricaemic participants (from B=9.8, 95% CI: 8.0-11.7, p<0.001 to B=7.3, 95% CI: 5.3-9.2, p<0.001) after controlling for TyG index. In the ascending segment, the relationship between SUA and FPG was partially mediated by TyG index in normouricaemic participants, but fully in hyperuricaemic participants. CONCLUSIONS: SUA had an inverted U-shaped relationship with FPG, and their positive relationship was fully mediated by insulin resistance in participants with hyperuricaemia but not those without.

Sensitive Oxidation of Sorbitol-Mediated Fe2+ by H2O2: A Reliable TD-NMR Method for Clinical Blood Glucose Detection.

The clinical challenge of high-accuracy blood glucose detection schemes is to overcome the detection error caused by the background interferences in different individuals. H2O2 as the specific product of glucose oxidation can be involved in the Fe2+/Fe3+ conversion and detected by the time-domain nuclear magnetic resonance (TD-NMR) method sensitively. But, in clinical applications, the oxidation of Fe2+ is susceptible to the complex sample substrates. In this work, we sorted out two kinds of possible interference mechanisms of Fe2+ oxidation in the NMR blood glucose detection method and proposed a feasible scheme that uses sorbitol to weaken the adverse effects of interference. We found that sorbitol-mediated Fe2+ can greatly enhance the sensitivity of the T2 value to H2O2. The chain reaction caused by sorbitol can significantly amplify the efficiency of Fe2+ oxidation at the same concentration of H2O2. Thereby, we can achieve the higher dilution multiple of serum samples to reduce the amount of interfering substances involved in the Fe2+/Fe3+ conversion. We justified the accuracy and availability of our method by successfully detecting and confirming the correlation between the T2 decrease and glucose concentration of the serum samples collected from 16 subjects. The sorbitol-Fe2+ glucose detection method with high sensitivity can be further combined with miniature NMR analyzers to satisfy the calibration requirements of glucose monitoring in diabetic patients instead of frequent medical visits.

Secondary-flow-aided single-train elastic-inertial focusing in low elasticity viscoelastic fluids.

Elastic-inertial focusing has attracted increasing interest in recent years due to the three-dimensional (3D) single-train focusing ability it offers. However, multi-train focusing, instead of single-train focusing, was observed in viscoelastic fluids with low elasticity as a result of the competition between inertia effect and viscoelasticity effect. To address this issue, we employed the secondary flow to facilitate single-train elastic-inertial focusing in low elasticity viscoelastic fluids. A three-section contraction-expansion channel was designed to induce the secondary flow to pinch the multiplex focusing trains into a single one exactly at the channel centerline. After demonstrating the focusing process and mechanism in our device, we systematically explored and discussed the effects of particle diameter, operational flow rate, polymer concentration, and channel dimension on particle focusing performances. Our device enables single-train focusing of particles in viscoelastic fluids with low elasticity, and offers advantages of planar single-layer structure, and sheathless, external-field free operation.

Inertial microfluidics for high-throughput cell analysis and detection: a review.

Since it was first proposed in 2007, inertial microfluidics has been extensively studied in terms of theory, design, fabrication, and application. In recent years, with the rapid development of microfabrication technologies, a variety of channel structures that can focus, concentrate, separate, and capture bioparticles or fluids have been designed and manufactured to extend the range of potential biomedical applications of inertial microfluidics. Due to the advantages of high throughput, simplicity, and low device cost, inertial microfluidics is a promising candidate for rapid sample processing, especially for large-volume samples with low-abundance targets. As an approach to cellular sample pretreatment, inertial microfluidics has been widely employed to ensure downstream cell analysis and detection. In this review, a comprehensive summary of the application of inertial microfluidics for high-throughput cell analysis and detection is presented. According to application areas, the recent advances can be sorted into label-free cell mechanical phenotyping, sheathless flow cytometric counting, electrical impedance cytometer, high-throughput cellular image analysis, and other methods. Finally, the challenges and prospects of inertial microfluidics for cell analysis and detection are summarized.

Combining Inertial Microfluidics with Cross-Flow Filtration for High-Fold and High-Throughput Passive Volume Reduction.

We reporte a three-stage spiral channel device for achieving high-fold and high-throughput passive volume reduction through coupling inertial microfluidics with cross-flow filtration. To understand the device physics and optimize the structure, the effects of critical channel design on particle dynamics and volume reduction performance were explored. Then the principle of volume reduction was used for concentrating cells from large-volume fluids, and the concentration performance of differently sized particles/cells in the determined device was quantitatively characterized over wide flow rates. The results indicated that our device could achieve high-efficiency cell concentration at a high throughput of over 4 mL/min. Finally, we successfully applied our device for the enrichment of rare tumor cells after being separated from the blood or peritoneal fluid and the extremely high fold concentration of white blood cells from the large-volume fluid. Using a serial concentration, an ultrahigh concentration fold of approximately 1100 could be achieved. Our device offers numerous advantages, such as high-processing throughput, high concentration fold, simple channel design, and low-cost fabrication. Thus, it holds the potential to be used as a sample concentration tool for disposable use in low-resource settings.

Ultrahigh throughput beehive-like device for blood plasma separation.

We report here a low-cost, rapid-prototyping, and beehive-like multilayer polymer microfluidic device for ultrahigh-throughput blood plasma separation. To understand the device physics and optimize the device structure, the effect of cross-sectional dimension and operational parameter on particle focusing behavior was explored using a single spiral microchannel device. Then, the blood plasma separation performance of the determined channel structure was validated using the blood samples with different hematocrits (HCTs). It was found that a high separation efficiency of 99% could be achieved using the blood sample with an HCT of 0.5% at a high throughput of 1 mL/min. Finally, a multilayer microfluidic device with a novel beehive-like multiplexing channel arrangement was developed for ultrahigh-throughput blood plasma separation. The prototype device could be fabricated within ∼1 hour utilizing the laser cutting and thermal lamination methods. The total processing throughput could reach up to 72 mL/min for 0.5% HCT sample with a plasma separation ratio close to 90%. Our device may hold potentials for the ultrahigh-throughput separation of blood plasma from large volume blood samples for downstream disease diagnosis.

Droplet-based microreactor for the production of micro/nano-materials.

Droplet-based microreactors are of great interest to researchers due to their incredible ability in the synthesis of micro/nano-materials with multi-function and complex geometry. In recent years, a broad range of micro/nano-materials has been synthesized in droplet-based microreactors, which provide apparent advantages, such as better reproducibility, reliable automation, and accurate manipulation. In this review, we give a comprehensive and in-depth insight into droplet-based microreactors, covering fundamental research from droplet generation and manipulation to the applications of droplet-based microreactors in micro/nano-material generation. We also explore the outlook for droplet-based microreactors and challenges that lie ahead and give a possible effort direction. We hope this review will promote communications among researchers and entrepreneurs.