Cell lysis methods for high-throughput screening or miniaturized assays

The widely and routinely used cell lysis techniques are still being evolved to address the technical needs of micro-scale sample processing, for example in high-throughput screening or miniaturized assays. This review focuses on several recently emerged mechanical, physical and biological methods designed for lysing cells on small scales for screening or analysis.     

Fluorescence polarization assays for high-throughput screening of neuropeptide FF receptors

We have developed the first fluorescence polarization assays of human neuropeptide FF2 receptors in 384-well microtiter plates. Assays are completed in a single well with no transfer, separation, or wash steps. The performance is suitable for high-throughput drug screening applications with regard to speed of analysis, magnitude of displaceable signal, precision, and sensitivity of various reagents. The rank order of potency of agonists and antagonists agrees well relative to the published radiometric filtration assays: DMe NPFF > NPFF > frog PP (Rana temporaria pancreatic polypeptide) > PQRFamide > BIBP 3226. The effect of highly colored compounds is very small on the polarization signal up to micromolar concentrations. The method serves as a simple and fast alternative to radioligand binding assays of antiobesity drug candidates related to NPFF receptors.     

膜片钳技术的新进展及其在药物高通量筛选中的应用

作为先进的细胞电生理技术,膜片钳一直被奉为研究离子通道的“金标准”。应用膜片钳技术可以证实细胞膜上离子通道的存在并能对其电生理特性、分子结构、药物作用机制等进行深入的研究。基因组学、蛋白质组学研究表明,以离子通道为靶标的药物研究在未来具有很大的发展空间。为了突破由于筛选技术所造成的针对离子通道为靶标的药物研发的瓶颈,近年来,对膜片钳技术进行了改进以适合药物高通量筛选的需求,由此产生了一些新的技术。本文就最近几年膜片钳技术的新进展及其在药物高通量筛选中的应用进行了综述。     

A microfluidic cell chip for virus isolation via rapid screening for permissive cells

Virus identification is a prerequisite not only for the early diagnosis of viral infectious diseases but also for the effective prevention of epidemics. Successful cultivation is the gold standard for identifying a virus, according to the Koch postulates. However, this requires screening for a permissive cell line, which is traditionally time-, reagent- and labor-intensive. Here, a simple and easy-to-operate microfluidic chip, formed by seeding a variety of cell lines and culturing them in parallel, is reported for use in virus cultivation and virus-permissive host-cell screening. The chip was tested by infection with two known viruses, enterovirus 71 (EV71) and influenza virus H1N1. Infection with EV71 and H1N1 caused significant cytopathic effects (CPE) in RD and MDCK cells, respectively, demonstrating that virus cultivation based on this microfluidic cell chip can be used as a substitute for the traditional plate-based culture method and reproduce the typical CPE caused by virus infection. Using this microfluidic cell chip method for virus cultivation could make it possible to identify an emerging virus in a high-throughput, automatic, and unprecedentedly fast way.     

用于药物筛选的微流控细胞阵列芯片

细胞区域分布培养以及如何有效地对微流体进行操控是微流控阵列芯片在细胞药物研究中的关键技术。本研究介绍了一种利用SU-8负性光刻胶模具和PDMS制作双层结构的微流控细胞阵列芯片的方法,该芯片通过C型的坝结构将进样细胞拦截在芯片的细胞培养的固定区域,键合双层PDMS构成阀控制层,阀网络的开关作用成功实现了芯片通道内微流体的操控,同时芯片设计了药物浓度梯度网络,产生6个不同浓度的药物刺激细胞。通过对芯片3种共培养细胞活性的检测和药物伊立替康(CTP-11)对肝癌细胞的浓度梯度刺激等实验结果验证该芯片在细胞研究和药物筛选等方面的可行性。     

Agonist-induced calcium response in single human platelets assayed in a microfluidic device

To facilitate drug discovery directed toward platelet-specific targets, we developed a platelet isolation and fluorophore-loading method that yields functionally responsive platelets in which we were able to detect agonist-induced calcium flux using a microfluidics-based screening platform. The platelet preparation protocol was designed to minimize preparation-induced platelet activation and to optimize signal strength. Measurement of platelet activation, as monitored by ratiometric determination of agonist-induced calcium flux in fluor-loaded human platelets, was optimized in a macrosample cuvette format in preparation for detection in a microfluidic chip-based assay. For the microfluidic device used in these studies, a cell density of 1 to 2 x 10(6) platelets per milliliter and a nominal flow rate of 5 to 10 nl per second provided optimal event resolution of 5 to 20 platelets traversing the detection volume per unit time. Platelets responded in a dose-dependent manner to adenosine diphosphate and protease-activating peptide (PAR) 1 thrombin receptor-activating peptide (TRAP). The work presented here constitutes proof-of-principle experiments demonstrating the enabling application of a microfluidic device to conduct high-throughput signaling studies and drug discovery screening against human platelet targets.     

Microfluidic three-dimensional cell culture of stem cells for high-throughput analysis

Although the recent advances in stem cell engineering have gained a great deal of attention due to their high potential in clinical research, the applicability of stem cells for preclinical screening in the drug discovery process is still challenging due to difficulties in controlling the stem cell microenvironment and the limited availability of high-throughput systems. Recently, researchers have been actively developing and evaluating three-dimensional (3D) cell culture-based platforms using microfluidic technologies, such as organ-on-a-chip and organoid-on-a-chip platforms, and they have achieved promising breakthroughs in stem cell engineering. In this review, we start with a comprehensive discussion on the importance of microfluidic 3D cell culture techniques in stem cell research and their technical strategies in the field of drug discovery. In a subsequent section, we discuss microfluidic 3D cell culture techniques for high-throughput analysis for use in stem cell research. In addition, some potential and practical applications of organ-on-a-chip or organoid-on-a-chip platforms using stem cells as drug screening and disease models are highlighted.     

Development and validation of high-throughput screening assays for poly(ADP-ribose) polymerase-2 inhibitors

Poly(ADP-ribose) polymerase-1 and -2 (PARP1/2) are two key facilitators of DNA repair and are implicated in the pathogenesis of cancers and several chronic diseases. Inhibitors of PARP1/2 have shown powerful therapeutic effects in the treatment of cancer, cerebral ischemia, and inflammation. In addition, evidence from several studies suggests unique functions for PARP2 in genome surveillance, spermatogenesis, adipogenesis, and T cell development, and PARP2-specific inhibitors might have many other applications. To acquire PARP1/2 inhibitors, many high-throughput screening (HTS) assays for PARP1 inhibitors have been developed. However, detailed screening assays for PARP2 inhibitors have not been reported. Herein, three HTS assays for PARP2 inhibitors were developed and validated with reference inhibitors in each case. The results suggest that the HTS assays for PARP2 inhibitors using chemical quantification of NAD⁺, biotin-based quantification of PAR, and ELISA quantification of PAR are sensitive, robust, and cost effective.     

毕赤酵母液滴微流控高通量筛选方法的建立与应用

巴斯德毕赤酵母是当前应用最为方便和广泛的外源蛋白表达系统之一,为了进一步提高其表达外源蛋白的能力,文中建立了基于液滴微流控的毕赤酵母高通量筛选方法,并以木聚糖酶融合荧光蛋白为例,筛选获得木聚糖酶表达和分泌能力提高的突变株。通过PCR扩增得到木聚糖酶xyn5基因和绿色荧光蛋白gfp基因融合片段,并克隆到毕赤酵母表达载体pPIC9K中构建出木聚糖酶融合绿色荧光蛋白的质粒pPIC9K-xyn5-gfp,电转化至毕赤酵母GS115中得到表达木聚糖酶和绿色荧光蛋白的毕赤酵母SG菌株。该菌株经过常压室温等离子体诱变后进行单细胞液滴包埋,液滴培养24h后进行微流控筛选,获得高表达木聚糖酶的突变菌株,进而用于下一轮的诱变突变库构建和筛选。以此类推,经过5轮液滴微流控筛选,获得一株高产菌株SG-m5,其木聚糖酶活为149.17U/mg,较出发菌株提升300%,分泌外源蛋白的能力较出发菌株提高160%。文中建立的毕赤酵母单细胞液滴微流控高通量筛选方法能达到每小时10万菌株的筛选通量,筛选百万级别的菌株库仅需10h,消耗荧光试剂体积100μL,对比传统的微孔板筛选方法降低试剂成本近百万倍,为高效、低成本筛选获得表达和分泌外源蛋白能力提高的毕赤酵母提供了一条新途径。     

A high-throughput screening assay for the carboxyltransferase subunit of acetyl-CoA carboxylase

One consequence of the dramatic rise of antibiotic-resistant pathogenic bacteria is the need for new targets for antibiotics. Because membrane lipid biogenesis is essential for bacterial growth, enzymes of the fatty acid biosynthetic pathway offer attractive possibilities for the development of new antibiotics. Acetyl-coenzyme A carboxylase (ACC) catalyzes the first committed and regulated step in fatty acid biosynthesis in bacteria and thus is a prime target for development of antibiotics. ACC is a multifunctional enzyme composed of three separate proteins. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin. The biotin carboxyl carrier protein features a biotin molecule covalently attached at Lys122 of the Escherichia coli enzyme. The carboxyltransferase subunit catalyzes the transfer of a carboxyl group from biotin to acetyl-coenzyme A (acetyl-CoA) to form malonyl-CoA. The objective of this study was to develop an assay for high-throughput screening for inhibitors of the carboxyltransferase subunit. The carboxyltransferase reaction was assayed in the reverse direction in which malonyl-CoA reacts with biocytin (an analog of the biotin carboxyl carrier protein) to form acetyl-CoA and carboxybiotin. The production of acetyl-CoA was coupled to citrate synthase, which produced citrate and coenzyme A. The amount of coenzyme A formed was detected using 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). The assay has been developed for use in both 96- and 384-well microplate formats and was validated using a known bisubstrate analog inhibitor of carboxyltransferase. The spectrophotometric readout in the visible absorbance range used in this assay does not generate the number of false negatives associated with frequently used NAD/NADH assay systems that rely on detection of NADH using UV absorbance.     

A high-throughput screening fluorescence polarization assay for fatty acid adenylating enzymes in Mycobacterium tuberculosis

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), encodes for an astonishing 34 fatty acid adenylating enzymes (FadDs), which play key roles in lipid metabolism. FadDs involved in lipid biosynthesis are functionally nonredundant and serve to link fatty acid and polyketide synthesis to produce some of the most architecturally complex natural lipids including the essential mycolic acids as well as the virulence-conferring phthiocerol dimycocerosates, phenolic glycolipids, and mycobactins. Here we describe the systematic development and optimization of a fluorescence polarization assay to identify small molecule inhibitors as potential antitubercular agents. We fluorescently labeled a bisubstrate inhibitor to generate a fluorescent probe/tracer, which bound with a K_D of 245 nM to FadD28. Next, we evaluated assay performance by competitive binding experiments with a series of known ligands and assessed the impact of control parameters including incubation time, stability of the signal, temperature, and DMSO concentration. As a final level of validation the LOPAC1280 library was screened in a 384-well plate format and the assay performed with a Z-factor of 0.75, demonstrating its readiness for high-throughput screening.     

Functional RNA microarrays for high-throughput screening of antiprotein aptamers

High-throughput methods for generating aptamer microarrays are described. As a proof-of-principle, the microarrays were used to screen the affinity and specificity of a pool of robotically selected antilysozyme RNA aptamers. Aptamers were transcribed in vitro in reactions supplemented with biotinyl-guanosine 5'-monophosphate, which led to the specific addition of a 5' biotin moiety, and then spotted on streptavidin-coated microarray slides. The aptamers captured target protein in a dose-dependent manner, with linear signal response ranges that covered seven orders of magnitude and a lower limit of detection of 1 pg/mL (70 fM). Aptamers on the microarray retained their specificity for target protein in the presence of a 10,000-fold (w/w) excess of T-4 cell lysate protein. The RNA aptamer microarrays performed comparably to current antibody microarrays and within the clinically relevant ranges of many disease biomarkers. These methods should also prove useful for generating other functional RNA microarrays, including arrays for genomic noncoding RNAs that bind proteins. Integrating RNA aptamer microarray production with the maturing technology for automated in vitro selection of antiprotein aptamers should result in the high-throughput production of proteome chips.     

SPR蛋白质芯片在输入性疟疾筛查中的应用

输入性疟疾已是我国疟疾防控的主要危险因素,如何对入境人员进行疟疾快速筛查是急需解决的难题。蛋白质芯片已被广泛应用于高通量筛选和诊断,本研究尝试构建了表面等离子共振技术 (Surface plasmon resonance,SPR) 蛋白芯片用于恶性疟疾的快速检测。采用聚乙二醇高分子处理的特异性吸附表面,以恶性疟疾特异性抗原富组氨酸蛋白Ⅱ (Histidine-rich protein Ⅱ,HRP2) 作为捕获探针,建立疟疾的微阵列芯片,并对芯片的最佳抗原固定浓度,检测的灵敏性和特异性,以及抗干扰能力进行了分析。该芯片可成功应用于恶性疟疾的筛查,具有无标记、即时快速的特点,与荧光定量PCR法相比,两种方法在敏感度和特异性方面无统计学差异。研究结果为一步研制疟疾分型鉴定蛋白质芯片奠定了基础,有利于对入境人员进行疟疾快速筛查。     

Application and prospects of high-throughput screening for in vitro neurogenesis

Over the past few decades, high-throughput screening (HTS) has made great contributions to new drug discovery. HTS technology is equipped with higher throughput, minimized platforms, more automated and computerized operating systems, more efficient and sensitive detection devices, and rapid data processing systems. At the same time, in vitro neurogenesis is gradually becoming important in establishing models to investigate the mechanisms of neural disease or developmental processes. However, challenges remain in generating more mature and functional neurons with specific subtypes and in establishing robust and standardized three-dimensional (3D) in vitro models with neural cells cultured in 3D matrices or organoids representing specific brain regions. Here, we review the applications of HTS technologies on in vitro neurogenesis, especially aiming at identifying the essential genes, chemical small molecules and adaptive microenvironments that hold great prospects for generating functional neurons or more reproductive and homogeneous 3D organoids. We also discuss the developmental tendency of HTS technology, e.g., so-called next-generation screening, which utilizes 3D organoid-based screening combined with microfluidic devices to narrow the gap between in vitro models and in vivo situations both physiologically and pathologically.     

A label-free microfluidic chip for the highly selective isolation of single and cluster CTCs from breast cancer patients

BackgroundCirculating tumor cells (CTCs) existing in peripheral blood can be used to predict the prognosis and survival of cancer patients. The study was designed to detect circulating tumor cells and circulating tumor single cell genes by applying microfluidic chip technology. It was used to explore the clinical application value in breast cancer.MethodsWe have developed a size-based CTCs sorting microfluidic chip, which contains a hexagonal array and a micro-pipe channel array to isolate and confirm both single CTCs and CTCs clusters. The sorting performance of the as-fabricated chip was tested by analyzing the clinical samples collected from 129 breast cancer patients and 50 healthy persons.ResultsIn this study, the chip can detect different immunophenotypes of CTCs in breast cancer patients. It was found that the new microfluidic device had high sensitivity (73.6%) and specificity (82.0%) in detecting CTCs. By detecting the blood samples of 129 breast cancer patients and 50 healthy blood donors, it was found that the number of CTCs was not associated with clinical factors such as age, gender, pathological type, and tumor size of breast cancer patients (P > 0.05), but was associated with TNM staging of breast cancer, with or without metastasis (P < 0.005). There was a statistically significant difference in the number of CTCs between luminal A (ER+/PR+/HER2-) and HER-2+ (ER-/PR-/HER2+) (P < 0.05). The best cut-off level distinguished by CTC between the breast cancer patients and the healthy persons was 3.5 cells/mL, with 0.845 for AUC-ROC, 0.790–0.901 for 95% CI, 73.6% for sensitivity, and 82% for specificity (P = 0.000). The combination of CTC, CEA, CA125 and CA153 can provide more effective breast cancer screening.ConclusionsThe CTCs analysis method presented here doesn''t rely on the specific antibody, such as anti-EpCAM, which would avoid the missed inspection caused by antibody-relied methods and offer more comprehensive biological information for clinical breast cancer diagnosis and treatment.     

Numerical design of a microfluidic chip for probing mechanical properties of cells

Microfluidic chips have been widely used to probe the mechanical properties of cells, which are recognized as a promising label-free biomarker for some diseases. In our previous work (Ye et al., 2018), we have studied the relationships between the transit time and the mechanical properties of a cell flowing through a microchannel with a single constriction, which potentially forms a basis for a microfluidic chip to measure cell’s mechanical properties. Here, we investigate this microfluidic chip design and examine its potential in performances. We first develop the simultaneous dependence of the transit time on both the shear and bending moduli of a cell, and then examine the chip sensitivity with respect to the cell mechanical properties while serializing a single constriction along the flow direction. After that, we study the effect of the flow velocity on the transit time, and also test the chip’s ability to identify heterogeneous cells with different mechanical properties. The results show that the microfluidic chip designed is capable of identifying heterogeneous cells, even when only one unhealthy cell is included. The serialization of chip can greatly increase the chip sensitivity with respect to the mechanical properties of cells. The flow with a higher velocity helps in not only promoting the chip throughput, but also in providing more accurate transit time measurements, because the cell prefers a symmetric deformation under a high velocity.     

A colorimetric assay optimization for high-throughput screening of dihydroorotase by detecting ureido groups

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine biosynthesis pathway and is a potential new antibacterial drug target. No target-based high-throughput screening (HTS) assay for this enzyme has been reported to date. Here, we optimized two colorimetric-based enzymatic assays that detect the ureido moiety of the DHOase substrate, carbamyl-aspartate (Ca-asp). Each assay was developed in a 40-μl assay volume using 384-well plates with a different color mix, diacetylmonoxime (DAMO)–thiosemicarbazide (TSC) or DAMO–antipyrine. The sensitivity and color interference of both color mixes were compared in the presence of common HTS buffer additives, including dimethyl sulfoxide, reducing agents, detergents, and bovine serum albumin. DAMO–TSC (Z′-factors 0.7–0.8) was determined to be superior to DAMO–antipyrine (Z′-factors 0.5–0.6) with significantly less variability within replicates. An HTS pilot screening with 29,552 compounds from four structurally diverse libraries confirmed the quality of our newly optimized colorimetric assay with DAMO–TSC. This robust method has no heating requirement, which was the main obstacle to applying previous assays to HTS. More important, this well-optimized HTS assay for DHOase, the first of its kind, should make it possible to screen large-scale compound libraries to develop new inhibitors against any enzymes that produce ureido functional groups.     

Development of a scintillation proximity binding assay for high-throughput screening of hematopoietic prostaglandin D2 synthase

Prostaglandin D₂ synthase (PGDS) catalyzes the isomerization of prostaglandin H₂ (PGH₂) to prostaglandin D₂ (PGD₂). PGD₂ produced by hematopoietic prostaglandin D₂ synthase (H-PGDS) in mast cells and Th2 cells is proposed to be a mediator of allergic and inflammatory responses. Consequently, inhibitors of H-PGDS represent potential therapeutic agents for the treatment of inflammatory diseases such as asthma. Due to the instability of the PGDS substrate PGH₂, an in-vitro enzymatic assay is not feasible for large-scale screening of H-PGDS inhibitors. Herein, we report the development of a competition binding assay amenable to high-throughput screening (HTS) in a scintillation proximity assay (SPA) format. This assay was used to screen an in-house compound library of approximately 280,000 compounds for novel H-PGDS inhibitors. The hit rate of the H-PGDS primary screen was found to be 4%. This high hit rate suggests that the active site of H-PGDS can accommodate a large diversity of chemical scaffolds. For hit prioritization, these initial hits were rescreened at a lower concentration in SPA and tested in the LAD2 cell assay. 116 compounds were active in both assays with IC₅₀s ranging from 6 to 807 nM in SPA and 82 nM to 10 μM in the LAD2 cell assay.     

Development of a high-throughput screening assay for the discovery of small-molecule SecA inhibitors

A major pathway for bacterial preprotein translocation is provided by the Sec-dependent preprotein translocation pathway. Proteins destined for Sec-dependent translocation are synthesized as preproteins with an N-terminal signal peptide, which targets them to the SecYEG translocase channel. The driving force for the translocation reaction is provided by the peripheral membrane ATPase SecA, which couples the hydrolysis of ATP to the stepwise transport of unfolded preproteins across the bacterial membrane. Since SecA is essential, highly conserved among bacterial species, and has no close human homologues, it represents a promising target for antibacterial chemotherapy. However, high-throughput screening (HTS) campaigns to identify SecA inhibitors are hampered by the low intrinsic ATPase activity of SecA and the requirement of hydrophobic membranes for measuring the membrane or translocation ATPase activity of SecA. To address this issue, we have developed a colorimetric high-throughput screening assay in a 384-well format, employing an Escherichia coli (E. coli) SecA mutant with elevated intrinsic ATPase activity. The assay was applied for screening of a chemical library consisting of ∼27,000 compounds and proved to be highly reliable (average Z′ factor of 0.89). In conclusion, a robust HTS assay has been established that will facilitate the search for novel SecA inhibitors.     

A new recombinant protein expression system for high-throughput screening in the yeast Yarrowia lipolytica

Development of a high-throughput eukaryotic screening procedure is important to increase success in obtaining improved enzymes through directed enzyme evolution. This procedure was developed for the yeast Yarrowia lipolytica which becomes the second eukaryotic host for this purpose. The extracellular lipase Lip2 was used as expressed enzyme but this system will be easily adjusted for other enzymes. We adapted and optimized the protocol for protein expression by Y. lipolytica in 96-well microplates. Yeast transformation efficiency and expression cassette insertion were increased by constructing a strain containing a zeta docking platform for targeted integration into the genome. The coefficient of variance of the full process was reduced from 36.3% to 18.9%. The main part of the variability (11.7%) arises from the specific lipase enzyme assay whereas the coefficient of variance concerning transformation, growth and expression steps represents only 7.2%. The rate of clone with no activity was reduced from 5.8% to 0.2%. Both transformation efficiency and variability are then compatible with high-throughput screening in the yeast Y. lipolytica.