High-throughput Device Research Articles

Autonomous high-throughput screening technology for accelerating drug molecule discovery and synthesis

<p indent="0mm">The discovery and synthesis of pharmaceutical small molecules is one of the important decisive steps in the process of new drug research and development. The traditional small molecule design and synthesis methods, however, is becoming increasingly difficult to meet the iteration and development speed of new drug molecules in modern human society. The recent emergence of chemical artificial intelligence and high-throughput screening (HTS) technology is a new direction to break through the bottleneck of traditional research and development efficiency. This review will focus on the development status of automatic HTS device driven by artificial intelligence decision algorithm, mainly including: (1) High-throughput screening device technology. Batch and flow HTS modes, reaction process intensification for HTS screening, and the analytical characterization technology for HTS are discussed. (2) Intelligent decision-making algorithm for driving HTS, discussing the autonomous decision algorithms for driving the automation device to realize intelligent autonomous exploration, target property optimization and high-quality experimental data generation. Finally, this review will discuss how to further develop intelligent HTS technology to accelerate the synthesis of pharmaceutical molecules in the future.

A simple high-throughput method for automated detection of Drosophila melanogaster light-dependent behaviours

BackgroundLike most living organisms, the fruit fly Drosophila melanogaster exhibits strong and diverse behavioural reactions to light. Drosophila is a diurnal animal that displays both short- and long-term responses to light, important for, instance, in avoidance and light wavelength preference, regulation of eclosion, courtship, and activity, and provides an important model organism for understanding the regulation of circadian rhythms both at molecular and circuit levels. However, the assessment and comparison of light-based behaviours is still a challenge, mainly due to the lack of a standardised platform to measure behaviour and different protocols created across studies. Here, we describe the Drosophila Interactive System for Controlled Optical manipulations (DISCO), a low-cost, automated, high-throughput device that records the flies’ activity using infrared beams while performing LED light manipulations.ResultsTo demonstrate the effectiveness of this tool and validate its potential as a standard platform, we developed a number of distinct assays, including measuring the locomotor response of flies exposed to sudden darkness (lights-off) stimuli. Both white-eyed and red-eyed wild-type flies exhibit increased activity after the application of stimuli, while no changes can be observed in Fmr1 null allele flies, a model of fragile X syndrome. Next, to demonstrate the use of DISCO in long-term protocols, we monitored the circadian rhythm of the flies for 48 h while performing an alcohol preference test. We show that increased alcohol consumption happens intermittently throughout the day, especially in the dark phases. Finally, we developed a feedback-loop algorithm to implement a place preference test based on the flies’ innate aversion to blue light and preference for green light. We show that both white-eyed and red-eyed wild-type flies were able to learn to avoid the blue-illuminated zones.ConclusionsOur results demonstrate the versatility of DISCO for a range of protocols, indicating that this platform can be used in a variety of ways to study light-dependent behaviours in flies.

Silica/Proteoliposomal Nanocomposite as a Potential Platform for Ion Channel Studies.

The nanostructuration of solid matrices with lipid nanoparticles containing membrane proteins is a promising tool for the development of high-throughput screening devices. Here, sol-gel silica-derived nanocomposites loaded with liposome-reconstituted KcsA, a prokaryotic potassium channel, have been synthesized. The conformational and functional stability of these lipid nanoparticles before and after sol-gel immobilization have been characterized by using dynamic light scattering, and steady-state and time-resolved fluorescence spectroscopy methods. The lipid-reconstituted KcsA channel entrapped in the sol-gel matrix retained the conformational and stability changes induced by the presence of blocking or permeant cations in the buffer (associated with the conformation of the selectivity filter) or by a drop in the pH (associated with the opening of the activation gate of the protein). Hence, these results indicate that this novel device has the potential to be used as a screening platform to test new modulating drugs of potassium channels.

Paper-based microfluidics: What can we expect?

Paper-based microfluidics: What can we expect?

Enabling the Internet of Bodies Through Capacitive Body Channel Access Schemes

The Internet of Bodies (IoB) is an imminent extension of the vast Internet of Things (IoT) domain, where wearable, ingestible, injectable, and implantable smart objects form a network in, on, and around the human body. The highly radiative nature of radio-frequency (RF) IoB devices unnecessarily extends the coverage range beyond the human body, which reduces energy efficiency, causes co-existence and interference issues, and exposes sensitive personal data to security threats. Alternatively, capacitive body channel communication (BCC) confine signal transmission to the human body to reduce signal leakage, experience less propagation loss, and reach pJ/b energy efficiency levels. Therefore, capacitive BCC is a key enabler to reach the ultimate design goals of ultra low power, high throughput, and small form-factor IoB devices. Albeit these attractive features, the communication and networking aspects of the capacitive BCC are not thoroughly explored yet. Therefore, this article proposes orthogonal and nonorthogonal capacitive body channel access schemes with or without cooperation among the IoB nodes. In order to address the Quality of Service (QoS) demand scenarios of different IoB applications, we present and formulate the max–min rate, max-sum rate, and QoS sufficient operational regimes, and then provide closed-form and numerical solution optimal power and phase time allocations. Extensive numerical results are analyzed to compare the performance of orthogonal and nonorthogonal schemes with and without cooperation for various design parameters under prescribed QoS regimes. The obtained results show that capacitive body channel access schemes can provide several Mb/s rates even at low transmission powers ranging between −60 and −90 dBm. Moreover, the cooperative schemes are shown to be effective to avoid performance degradation caused by increasing network size, low transmission power, and poor channel quality.

MINI: A high-throughput point-of-care device for performing hundreds of nucleic acid tests per day

There are a variety of infectious diseases with a high incidence and mortality in limited resource settings that could benefit from rapid point of care molecular diagnosis. Global health efforts have sought to implement mass-screening programs to provide earlier detection and subsequent treatment in an effort to control transmission and improve health outcomes. However, many of the current diagnostic technologies under development are limited to fewer than 10 samples per run, which inherently restricts the screening throughput of these devices. We have developed a high throughput device called “MINI” that is capable of testing hundreds of samples per day at the point-of-care. MINI can utilize multiple energy sources – electricity, flame, or solar – to perform loop-mediated isothermal amplification (LAMP) in a portable and robust device which is ideal for use in limited resource settings. The unique opto-electronic design of MINI minimizes the energy and space requirements of the device and maximizes the optical isolation and signal clarity, enabling point-of-care analysis of 96 unique samples at once. We show comparable performance to a commercial instrument using two different LAMP assays for Kaposi's sarcoma-associated herpesvirus and a common housekeeping gene, GAPDH. With a single device capable of running hundreds of samples per day, increased access to modern molecular diagnostics could improve health outcomes for a variety of diseases common in limited resource settings.

A high-throughput microfluidic device based on controlled incremental filtration to enable centrifugation-free, low extracorporeal volume leukapheresis

Leukapheresis, the extracorporeal separation of white blood cells (WBCs) from red blood cells (RBCs) and platelets (PLTs), is a life-saving procedure used for treating patients with cancer and other conditions, and as the initial step in the manufacturing of cellular and gene-based therapies. Well-tolerated by adults, leukapheresis poses a significant risk to neonates and low-weight infants because the extracorporeal volume (ECV) of standard centrifugation-based machines represents a particularly large fraction of these patients’ total blood volume. Here we describe a novel high-throughput microfluidic device (with a void volume of 0.4 mL) based on controlled incremental filtration (CIF) technology that could replace centrifugation for performing leukapheresis. The CIF device was tested extensively using whole blood from healthy volunteers at multiple hematocrits (5–30%) and flow rates (10–30 mL/min). In the flow-through regime, the CIF device separated WBCs with > 85% efficiency and 10–15% loss of RBCs and PLTs while processing whole blood diluted with saline to 10% hematocrit at a flow rate of 10 mL/min. In the recirculation regime, the CIF device demonstrated a similar level of separation performance, virtually depleting WBCs in the recirculating blood (~ 98% reduction) by the end of a 3.5-hour simulated leukapheresis procedure. Importantly, the device operated without clogging or decline in separation performance, with minimal activation of WBCs and PLTs and no measurable damage to RBCs. Compared to the typical parameters of centrifugation-based leukapheresis, the CIF device had a void volume at least 100-fold smaller, removed WBCs about twice as fast, and lost ~ 2–3-fold fewer PLTs, while operating at a flow rate compatible with the current practice. The hematocrit and flow rate at which the CIF device operated were significantly higher than previously published for other microfluidic cell separation methods. Finally, this study is the first to demonstrate a highly efficient separation of cells from recirculating blood using a microfluidic device. Overall, these findings suggest the feasibility of using high-throughput microfluidic cell separation technology to ultimately enable centrifugation-free, low-ECV leukapheresis. Such a capability would be particularly useful in young children, a vulnerable group of patients who are currently underserved.

Effects of working pressure on the material and defect properties of Sb2S3 thin-film solar cells achieved by the VTD method.

Antimony sulfide (Sb2S3), an emerging material for photovoltaic devices, has drawn growing research interest due to its inexpensive and high-throughput device production. In this study, the material and defect properties of Sb2S3 thin films prepared by the vapor transport deposition (VTD) method at different working pressures were studied. Solar cells based on a structure of glass/ITO/CdS/Sb2S3/Au were fabricated. The working pressure showed a significant effect on the device's performance. The current density versus voltage measurement and scanning electron microscopy analysis outcome were utilized to investigate the photovoltaic and microstructural properties in the samples. The compositional analysis by energy dispersive X-ray spectroscopy measurement confirmed the Sb/S ratio as 2:2.8 for the thin films. The identification and characterization of the defects present in Sb2S3 thin films were performed via admittance measurements. Compared to the defect density, the defect energy level was found to inherit a more important role in the device's performance. The best solar cell performance with better crystal quality, lower defect density, and longer capture lifetime was achieved under the substrate working pressure of 2 Pa. The highest efficiency was found to be 0.86% with Voc=0.55V, Jsc=5.07mA/cm2.

Multiplexed high-throughput localized electroporation workflow with deep learning-based analysis for cell engineering.

Manipulation of cells for applications such as biomanufacturing and cell-based therapeutics involves introducing biomolecular cargoes into cells. However, successful delivery is a function of multiple experimental factors requiring several rounds of optimization. Here, we present a high-throughput multiwell-format localized electroporation device (LEPD) assisted by deep learning image analysis that enables quick optimization of experimental factors for efficient delivery. We showcase the versatility of the LEPD platform by successfully delivering biomolecules into different types of adherent and suspension cells. We also demonstrate multicargo delivery with tight dosage distribution and precise ratiometric control. Furthermore, we used the platform to achieve functional gene knockdown in human induced pluripotent stem cells and used the deep learning framework to analyze protein expression along with changes in cell morphology. Overall, we present a workflow that enables combinatorial experiments and rapid analysis for the optimization of intracellular delivery protocols required for genetic manipulation.

Prediction and Observation of Robust One-Way Bulk States in a Gyromagnetic Photonic Crystal

We report a two-dimensional heterogeneous Haldane model composed of alternately stacking modified Haldane lattices with opposite next-nearest-neighbor hoppings, and predict the emergence of robust one-way bulk states by an abinitio theoretical calculation. These unique bulk states transport unidirectionally and are robust against backscattering from impurities in the strip bulk. By analogy with the heterogeneous Haldane model, we further confirm by numerical simulations and experimental measurements the existence of robust one-way bulk states in a two-dimensional microwave gyromagnetic photonic crystal, and demonstrate their robust one-way property over a long-distance even in the presence of metallic obstacles. Our study provides the strong support for the generalization and application of band theories to fermionic and bosonic systems, and paves a way for the implementation of high-throughput robust energy transmission materials and devices.

Abstract 6031: Microfluidics: Simulating the gut microbiome for early cancer detection

Abstract Background: Cancer is the second leading cause of death globally and ~39.5% of people will be diagnosed with cancer at some point during their lifetimes. Thus, there is an unmet need to identify novel strategies for early cancer detection and prevention. The emerging evidence suggests that the gut microbiome has a role in promoting cancer. This microbiome including bacteria plays a vital role in maintaining homeostasis in the body. An imbalance in bacterial composition may cause diseases including cancer. Here we developed a microfluidic chip that can accurately simulate the gut microbiome to test the effects of bacteria and therapies on cancer cells. Methods and Results: To test the causal effect of bacteria on cancer, we developed a new high-throughput microfluidic device for simulating the environment of the gut. Initially, we used the photolithography technique where we designed the chip in AutoCAD and fabricated using photoresist resins and Polydimethylsiloxane (PDMS). Next, we tested the effect of bacteria on the growth of colorectal cancer cells. For this, we cultured colorectal cancer cells (HCT-116) with lipopolysaccharide (LPS), which is found in the outer membrane of bacteria, as well as the Bacillus bacteria in our microfluidics. Our data show that both LPS and Bacillus significantly accelerate the growth of cancer cells 2.02 times (p value = 0.012) and 1.58 times (p value = 0.011), respectively, over a 4 day culture period. These results show that the increased presence of certain bacteria can promote cancer cell growth and that our chip can be used to test the specific correlation between bacteria and cancer cell growth. The previously described method was inefficient and time-consuming. To overcome this limitation, we designed a new chip that allows running 16 samples at once with improved efficiency and accuracy. The template of the device that had 16 microfluidic channels was printed by a 3D printer and used for PDMS replica molding. The PDMS device was attached to the modified multiwell plate to feed media to and collect waste from each channel in a high-throughput manner. In the initial design, the bacteria grew faster than cancer cells taking over the chips. Our new design has dual layered chambers to keep bacteria and cancer cells separated by a membrane, allowing only bacterial secretions to pass through the membrane to cancer cells, mimicking the human gut. The new design also allowed the chip to maintain continuous microfluidic flow and a hypoxic environment. Conclusion: Our research demonstrates that the new microfluidic device has broader implications including simulating other body organs such as the lung and liver, and testing the impact of viruses such as influenza and COVID-19 on human cells. This device can be used to test both the effect of bacteria and new treatment on clinical samples for the identification of personalized therapy, thus reducing the need for mouse model testing, which is a lengthy and expensive process. Citation Format: Ekansh Mittal, Young Bock Kang. Microfluidics: Simulating the gut microbiome for early cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6031.

Abstract 2279: Cas9 based targeted nanopore sequencing helps identify structural variants in breast cancer genes

Abstract Structural variations (SV), a hallmark of genomic instability in cancer, tend to be recurrent &amp; have been associated with several cancer types wherein they either activate oncogenes or inactivate tumor suppressor genes. NGS is mostly blind to large SVs, lacking sensitivity with FPR up to 89% in SV detection. Long-read sequencing (LRS) can address larger variations by generating read lengths of tens of thousands of bases &amp; have helped identify thousands of genomic features pertinent to cancer previously missed by NGS. However, throughput &amp; coverage of whole genome LRS makes it infeasible to conduct large-scale genomic studies to detect rare alleles. Cancer cells within the same sample can be heterogenous, with subpopulations exhibiting different genomic features, that are difficult to detect with just 30x whole genome sequencing. Targeted sequencing significantly improves accuracy &amp; coverage by offering depths necessary to detect these rare alleles in a heterogenous population of cells. Recently, we developed ACME, an Affinity-based Cas9-Mediated Enrichment approach that is an improvement on nanopore Cas9-targeted sequencing (nCATS) from Oxford Nanopore Technologies with the inclusion of a background reduction step. We targeted 10 prominent cancer genes in MCF 10A &amp; SK-BR-3 breast cell lines with ACME &amp; observed an increase in enrichment &amp; coverage of all genes on the panel, with enrichment as high as 5000-fold for some genes. We achieved a ~ 75-fold enrichment &amp; 35-65x coverage of the BRCA2 region, an important breast cancer gene that was a ~90 kb target on our panel. Across our panel, we found that ACME helps increase the number of single contiguous reads that span the entire target, which ultimately helps with better alignment &amp; SV detection. ACME detected all SVs within our target regions that had been previously inferred by PacBio &amp; ONT whole-genome LRS, but with higher depth. This allows for rare variant detection, making it an effective long-read targeting platform. We are currently developing ACME + native barcoding, which gave us mean target coverage of 10-30x in initial testing, to enable sample multiplexing along with target multiplexing. Our efforts are also directed towards expanding our cancer gene panel to target 35 genes common between breast, pancreatic, &amp; colorectal cancer. We have also successfully expanded ACME’s use to the PromethION high throughput device (currently unsupported by ONT for nCATS) &amp; have observed a 4-fold increase in coverage when compared to GridION using the same sample and mass for library prep. Performing high throughput targeted LRS on &amp;gt;15 samples per PromethION flowcell would allow SV analysis of several important cancer genes across 100s of samples, helping define the landscape of such variants in the population &amp; identify regions of therapeutic or diagnostic interest. Citation Format: Shruti V. Iyer, Melissa Kramer, Sara Goodwin, W. Richard McCombie. Cas9 based targeted nanopore sequencing helps identify structural variants in breast cancer genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2279.

Experimental and Modeling Based Investigations of Process Parameters on a Novel, 3D Printed and Self-Insulated 24-Well, High-Throughput 3D Microelectrode Array Device for Biological Applications

We have investigated the effect of printing angle and device orientation on the development of high-throughput (HT), self-insulated, 3D printed 3D Microelectrode Arrays (HT-3DMEAs) for <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">in vitro</i> biotechnology applications and its repercussions for optical, mechanical, and electrical properties of the final device. Makerspace based development using micro-Stereolithography ( <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\boldsymbol {\mu }$ </tex-math></inline-formula> SLA) 3D printing and ink casting technique were carried out to develop a 24-well device with self-insulated 3D microelectrodes, with <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">100% yield and 7 microtowers per well</i> acting as supports for the microelectrodes. These MEAs were further characterized by Optical Microscopy, Electrical Impedance Spectroscopy (EIS), Laser Scanning Confocal Microscopy, Scanning Electron Microscopy (SEM), and UV-Vis Spectroscopy. Surface flatness required for the microelectrode formation was obtained for 0° tilt printed version at top-side orientation. The process optimization and results were interpreted using established <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\boldsymbol {\mu }$ </tex-math></inline-formula> SLA theory and COMSOL modeling. Artifact size determination with a maximum 6% error was predicted. Experimental finding on the microelectrode formation, i.e., a potentially useful microbullet shape was observed and confirmed using Laser Confocal and SEM microscopy characterization and COMSOL simulation. These results allow for greater understanding of the device-process relationships for HT-3DMEAs and paves the pathway towards scalability, denser electrodes and even higher well counts for HT screening in biotechnology. [2021-0201]

Machine Learning in the Analysis of Multispectral Reads in Maize Canopies Responding to Increased Temperatures and Water Deficit

Real-time monitoring of crop responses to environmental deviations represents a new avenue for applications of remote and proximal sensing. Combining the high-throughput devices with novel machine learning (ML) approaches shows promise in the monitoring of agricultural production. The 3 × 2 multispectral arrays with responses at 610 and 680 nm (red), 730 and 760 nm (red-edge) and 810 and 860 nm (infrared) spectra were used to assess the occurrence of leaf rolling (LR) in 545 experimental maize plots measured four times for calibration dataset (n = 2180) and 145 plots measured once for external validation. Multispectral reads were used to calculate 15 simple normalized vegetation indices. Four ML algorithms were assessed: single and multilayer perceptron (SLP and MLP), convolutional neural network (CNN) and support vector machines (SVM) in three validation procedures, which were stratified cross-validation, random subset validation and validation with external dataset. Leaf rolling occurrence caused visible changes in spectral responses and calculated vegetation indexes. All algorithms showed good performance metrics in stratified cross-validation (accuracy &gt;80%). SLP was the least efficient in predictions with external datasets, while MLP, CNN and SVM showed comparable performance. Combining ML with multispectral sensing shows promise in transition towards agriculture based on data-driven decisions especially considering the novel Internet of Things (IoT) avenues.

Biophysical studies of cancer cells' traverse-vessel behaviors under different pressures revealed cells' motion state transition.

Circulating tumor cells (CTCs) survive in the bloodstream and then seed and invade to foster tumor metastasis. The arrest of cancer cells is favored by permissive flow forces and geometrical constraints. Through the use of high-throughput microfluidic devices designed to mimic capillary-sized vessels, we applied pressure differences to cancer cells (MCF-7 cell line) and recorded the cell traverse-vessel behaviors. Our results showed that cancer cells transform from a Newtonian droplet state to an adhesion/migration state when cancer cells traverse artificial vessels. To explain these phenomena, a modified Newtonian droplet model was also proposed. These phenomena and the modified model may reveal how CTCs in the blood seed and invade vessels under suitable conditions.

Real-time 96-well optoelectronic micro plate for kinetic and stability investigation of cytochrome P450 BM3

Simultaneous optical and electrochemical measurements are advantageous for biosensor technology. Although this is easy for single samples, parallelization is challenging, when ensuring spatial and temporal compatibility of measurement techniques, especially if the device is to be integrated into existing laboratory routines. Here, we developed an electrochemical-optical 96-well micro plate (eco-MP) to track the bioelectrocatalysis of cytochrome P450 BM3 fluorometrically and chronoamperometrically in a simultaneous and real-time manner. Fluorometric readout enables unbiased quantitative determination of the enzymatic product resorufin in parallel to the chronoamperometric data acquisition. We implemented 96 miniaturized three-electrode setups in the eco-MP with a 3D-printed divided cell architecture leading to 192 half-cells. Using a conventional fluorescence reader we demonstrate the potential of the developed eco-MP to control and monitor the active state of cytochrome P450 BM3 over hours. The benefit of reactive oxygen species scavenging enzymes on bioelectrocatalytic activity of P450 BM3 could be time-resolved assessed. We applied the eco-MP also to investigate the tolerance of P450 BM3 to organic solvent dimethyl sulfoxide, as an example of an area in bioelectrocatalysis where high-throughput measurement devices will kick in. Overall, this work stresses the demand for convenient multiparametric sensor technology in the growing field of electrified interfaces.

A two-stage approach for the spatio-temporal analysis of high-throughput phenotyping data

High throughput phenotyping (HTP) platforms and devices are increasingly used for the characterization of growth and developmental processes for large sets of plant genotypes. Such HTP data require challenging statistical analyses in which longitudinal genetic signals need to be estimated against a background of spatio-temporal noise processes. We propose a two-stage approach for the analysis of such longitudinal HTP data. In a first stage, we correct for design features and spatial trends per time point. In a second stage, we focus on the longitudinal modelling of the spatially corrected data, thereby taking advantage of shared longitudinal features between genotypes and plants within genotypes. We propose a flexible hierarchical three-level P-spline growth curve model, with plants/plots nested in genotypes, and genotypes nested in populations. For selection of genotypes in a plant breeding context, we show how to extract new phenotypes, like growth rates, from the estimated genotypic growth curves and their first-order derivatives. We illustrate our approach on HTP data from the PhenoArch greenhouse platform at INRAE Montpellier and the outdoor Field Phenotyping platform at ETH Zürich.

A high-throughput microfluidic device inspired by the Wheatstone bridge principle for characterizing the mechanical properties of single cells.

The mechanical properties of single cells have been recognized as biomarkers for identifying individual cells and diagnosing human diseases. Microfluidic devices based on the flow cytometry principle, which are not limited by the vision field of a microscope and can achieve a very high throughput, have been extensively adopted to measure the mechanical properties of single cells. However, these kinds of microfluidic devices usually required pressure-driven pumps with a very low flow rate and high precision. In this study, we developed a high-throughput microfluidic device inspired by the Wheatstone bridge principle for characterizing the mechanical properties of single cells. The microfluidic analogue of the Wheatstone bridge not only took advantage of flow cytometry, but also allowed precise control of a very low flow rate through the constricted channel with a higher input flow rate generated by a commercially available pressure-driven pump. Under different input flow rates of the pump, the apparent elastic moduli and the fluidity of osteosarcoma (U-2OS) cells and cervical carcinoma (HeLa) cells were measured by monitoring their dynamic deformations passing through the bridge-channel with different sizes of rectangular constrictions. The results showed that the input flow rate had little effect on measuring the mechanical properties of the cells, while the ratio of cell radius to effective constriction radius was different, i.e., for U-2OS cells it was 1.20 and for HeLa cells it was 1.09. Under this condition compared with predecessors, our statistic results of cell mechanical properties exhibited minimal errors. Furthermore, the cell viability after measurements was kept above 90% that demonstrated the non-destructive property of our proposed method.

Stator Dynamics Depending on Sodium Concentration in Sodium-Driven Bacterial Flagellar Motors.

Bacterial flagellar motor (BFM) is a large membrane-spanning molecular rotary machine for swimming motility. Torque is generated by the interaction between the rotor and multiple stator units powered by ion-motive force (IMF). The number of bound stator units is dynamically changed in response to the external load and the IMF. However, the detailed dynamics of stator unit exchange process remains unclear. Here, we directly measured the speed changes of sodium-driven chimeric BFMs under fast perfusion of different sodium concentration conditions using computer-controlled, high-throughput microfluidic devices. We found the sodium-driven chimeric BFMs maintained constant speed over a wide range of sodium concentrations by adjusting stator units in compensation to the sodium-motive force (SMF) changes. The BFM has the maximum number of stator units and is most stable at 5 mM sodium concentration rather than higher sodium concentration. Upon rapid exchange from high to low sodium concentration, the number of functional stator units shows a rapidly excessive reduction and then resurrection that is different from predictions of simple absorption model. This may imply the existence of a metastable hidden state of the stator unit during the sudden loss of sodium ions.

Tunning Intermolecular Interaction of Peptide-Conjugated AIEgen in Nano-Confined Space for Quantitative Detection of Tumor Marker Secreted from Cells.

Determining the expression level of biomarkers is crucial for disease diagnosis. However, the low abundance of biomarkers in the early stage makes the detection extremely difficult by traditional aggregation-induced emission (AIE)-based fluorescent probes. Here, by tuning the intermolecular interaction, a two steps-based MP/NPs-SLIPS sensing system is designed for ultrasensitive detection of the tumor marker matrix metalloproteinase-2 (MMP-2). During the sensing process, aggregation of AIE residual could be intensified through the electrostatic absorption by negatively charged nanoparticles (NPs), as well as the confined space formed by the self-assembly of NPs to photonic crystals (PCs) on slippery lubricant-infused porous substrates (SLIPS). The fluorescent signals obviously increased with a strengthened aggregation degree, which contributes to improved sensitivity. Thus, the limit of detection is decreased to 3.7 ng/mL for MP/NPs-SLIPS sensing system, which could be used for detecting the MMP-2 secreted by tumor cells directly. This strategy also demonstrated its potential applications as high-throughput detection devices and will be of significance for the ultrasensitive analysis of biomarkers.