Lab‐on‐a‐Chip‐Based High‐Throughput Screening of the Genotoxicity of Engineered Nanomaterials

The continuous increasing of engineered nanomaterials (ENMs) in our environment, their combinatorial diversity, and the associated genotoxic risks, highlight the urgent need to better define the possible toxicological effects of ENMs. In this context, we present a new high‐throughput screening (HTS) platform based on the cytokinesis‐block micronucleus (CBMN) assay, lab‐on‐chip cell sorting, and automated image analysis. This HTS platform has been successfully applied to the evaluation of the cytotoxic and genotoxic effects of silver nanoparticles (AgNPs) and silica nanoparticles (SiO₂NPs). In particular, our results demonstrate the high cyto‐ and genotoxicity induced by AgNPs and the biocompatibility of SiO₂NPs, in primary human lymphocytes. Moreover, our data reveal that the toxic effects are also dependent on size, surface coating, and surface charge. Most importantly, our HTS platform shows that AgNP‐induced genotoxicity is lymphocyte sub‐type dependent and is particularly pronounced in CD2+ and CD4+ cells.     

Mass spectrometric techniques for label-free high-throughput screening in drug discovery

High-throughput screening (HTS) is an important tool for finding active compounds to initiate medicinal chemistry programs in pharmaceutical discovery research. Traditional HTS methods rely on fluorescent or radiolabeled reagents and/or coupling assays to permit quantitation of enzymatic target inhibition or activation. Mass spectrometry-based high-throughput screening (MS-HTS) is an alternative that is not susceptible to the limitations imposed by labeling and coupling enzymes. MS-HTS offers a selective and sensitive analytical method for unlabeled substrates and products. Furthermore, method development times are reduced without the need to incorporate labels or coupling assays. MS-HTS also permits screening of targets that are difficult or impossible to screen by other techniques. For example, enzymes that are challenging to purify can lead to the nonspecific detection of structurally similar components of the impure enzyme or matrix of membraneous enzymes. The high selectivity of tandem mass spectrometry (MS/MS) enables these screens to proceed with low levels of background noise to sensitively discover interesting hits even with relatively weak activity. In this article, we describe three techniques that we have adapted for large-scale (approximately 175,000 sample) compound library screening, including four-way parallel multiplexed electrospray liquid chromatography tandem mass spectrometry (MUX-LC/MS/MS), four-way parallel staggered gradient liquid chromatography tandem mass spectrometry (LC/MS/MS), and eight-way staggered flow injection MS/MS following 384-well plate solid-phase extraction (SPE). These methods are capable of analyzing a 384-well plate in 37 min, with typical analysis times of less than 2 h. The quality of the MS-HTS approach is demonstrated herein with screening data from two large-scale screens.     

Visualizing Human Telomerase Activity with Primer‐Modified Au Nanoparticles

Telomerase is over‐expressed in over 85% of all known human tumors. This renders the enzyme a valuable biomarker for cancer diagnosis and an important therapeutic target. The most widely used telomeric repeat amplification protocol (TRAP) assay has been questioned for telomerase detection. It is reported that human telomerase activity can be visualized by using primer‐modified Au nanoparticles. The working principle is based on the elongated primers conjugated to the gold nanoparticle (AuNP) surface, which can fold into a G‐quadruplex to protect the AuNPs from the aggregation. The developed simple and sensitive colorimetric assay can measure telomerase activity down to 1 HeLa cell µL^?1. More importantly, this assay can be easily extended to high‐throughput and automatic format. The AuNP‐TS method is PCR‐free and therefore avoids the amplification‐related errors and becomes more reliable to evaluate telomerase activity. This assay has also been used for initial screening of telomerase inhibitors as anticancer drug agents.     

Assays of endogenous caspase activities: a comparison of mass spectrometry and fluorescence formats

This paper describes a label-free assay for measuring endogenous caspase protease activities in cell lysates. The assay format, termed SAMDI-MS (self-assembled monolayers for matrix assisted laser desorption ionization time-of-flight mass spectrometry), is based on the enzymatic modification of peptides immobilized to monolayer substrates, followed by direct detection of the products with mass spectrometry. Monolayers presenting peptide substrates for either caspase-3 or -8 were treated with lysates from Jurkat cells that were stimulated with staurosporine and SKW6.4 cells that were stimulated with LzCD95L. In both cases, the SAMDI assays reported on the activation of endogenous caspase enzymes with levels of detection that are similar to those observed using the commonly employed fluorogenic assays. The use of longer peptide substrates, which are not compatible with the fluorogenic assays, provided for a better resolution of the two caspase activities. This work is significant because it demonstrates that the SAMDI assay can be used to measure endogenous enzyme activities and because it avoids the loss of activity and specificity that often accompany label-dependent assay formats.     

A Microfabricated Sandwiching Assay for Nanoliter and High‐Throughput Biomarker Screening

Cells secrete substances that are essential to the understanding of numerous immunological phenomena and are extensively used in clinical diagnoses. Countless techniques for screening of biomarker secretion in living cells have generated valuable information on cell function and physiology, but low volume and real‐time analysis is a bottleneck for a range of approaches. Here, a simple, highly sensitive assay using a high‐throughput micropillar and microwell array chip (MIMIC) platform is presented for monitoring of biomarkers secreted by cancer cells. The sensing element is a micropillar array that uses the enzyme‐linked immunosorbent assay (ELISA) mechanism to detect captured biomolecules. When integrated with a microwell array where few cells are localized, interleukin 8 (IL‐8) secretion can be monitored with nanoliter volume using multiple micropillar arrays. The trend of cell secretions measured using MIMICs matches the results from conventional ELISA well while it requires orders of magnitude less cells and volumes. Moreover, the proposed MIMIC is examined to be used as a drug screening platform by delivering drugs using micropillar arrays in combination with a microfluidic system and then detecting biomolecules from cells as exposed to drugs.     

Graphene Oxide‐Assisted and DNA‐Modulated SERS of AuCu Alloy for the Fabrication of Apurinic/Apyrimidinic Endonuclease 1 Biosensor

Fabrication of high‐performance surface‐enhanced Raman scattering (SERS) biosensors relies on the coordination of SERS substrates and sensing strategies. Herein, a SERS active AuCu alloy with a starfish‐like structure is prepared using a surfactant‐free method. By covering the anisotropic AuCu alloy with graphene oxide (GO), enhanced SERS activity is obtained owing to graphene‐enhanced Raman scattering and assembly of Raman reporters. Besides, stability of SERS is promoted based on the protection of GO to the AuCu alloy. Meanwhile, it is found that SERS activity of AuCu/GO can be regulated by DNA. The regulation is sequence and length dual‐dependent, and short polyT reveals the strongest ability of enhancing the SERS activity. Relying on this phenomenon, a SERS biosensor is designed to quantify apurinic/apyrimidinic endonuclease 1 (APE1). Because of the APE1‐induced cycling amplification, the biosensor is able to detect APE1 sensitively and selectively. In addition, APE1 in human serum is analyzed by the SERS biosensor and enzyme‐linked immunosorbent assay (ELISA). The data from the SERS method are superior to that from ELISA, indicating great potential of this biosensor in clinical applications.     

Temporal Sampling of Enzymes from Live Cells by Localized Electroporation and Quantification of Activity by SAMDI Mass Spectrometry

Measuring changes in enzymatic activity over time from small numbers of cells remains a significant technical challenge. In this work, a method for sampling the cytoplasm of cells is introduced to extract enzymes and measure their activity at multiple time points. A microfluidic device, termed the live cell analysis device (LCAD), is designed, where cells are cultured in microwell arrays fabricated on polymer membranes containing nanochannels. Localized electroporation of the cells opens transient pores in the cell membrane at the interface with the nanochannels, enabling extraction of enzymes into nanoliter‐volume chambers. In the extraction chambers, the enzymes modify immobilized substrates, and their activity is quantified by self‐assembled monolayers for matrix‐assisted laser desorption/ionization (SAMDI) mass spectrometry. By employing the LCAD‐SAMDI platform, protein delivery into cells is demonstrated. Next, it is shown that enzymes can be extracted, and their activity measured without a loss in viability. Lastly, cells are sampled at multiple time points to study changes in phosphatase activity in response to oxidation by hydrogen peroxide. With this unique sampling device and label‐free assay format, the LCAD with SAMDI enables a powerful new method for monitoring the dynamics of cellular activity from small populations of cells.     

Continuous flow immobilized enzyme reactor-tandem mass spectrometry for screening of AChE inhibitors in complex mixtures

A method is described for identifying bioactive compounds in complex mixtures based on the use of capillary-scale monolithic enzyme-reactor columns for rapid screening of enzyme activity. A two-channel nanoLC system was used to continuously infuse substrate coupled with automated injections of substrate/small molecule mixtures, optionally containing the chromogenic Ellman reagent, through sol-gel derived acetylcholinesterase (AChE) doped monolithic columns. This is the first report of AChE encapsulated in monolithic silica for use as an immobilized enzyme reactor (IMER), and the first use of such IMERs for mixture screening. AChE IMER columns were optimized to allow rapid functional screening of compound mixtures based on changes in the product absorbance or the ratio of mass spectrometric peaks for product and substrate ions in the eluent. The assay had robust performance and produced a Z' factor of 0.77 in the presence of 2% (v/v) DMSO. A series of 52 mixtures consisting of 1040 compounds from the Canadian Compound Collection of bioactives was screened and two known inhibitors, physostigmine and 9-aminoacridine, were identified from active mixtures by manual deconvolution. The activity of the compounds was confirmed using the enzyme reactor format, which allowed determination of both IC(50) and K(I) values. Screening results were found to correlate well with a recently published fluorescence-based microarray screening assay for AChE inhibitors.     

A Single‐Cell Assay for Time Lapse Studies of Exosome Secretion and Cell Behaviors

To understand the inhomogeneity of cells in biological systems, there is a growing demand on the capability of characterizing the properties of individual single cells. Since single‐cell studies require continuous monitoring of the cell behaviors, an effective single‐cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single‐cell technologies cannot provide proper environments to sustain cell growth and, proliferation of single cells and convenient, noninvasive tests of single‐cell behaviors from molecular markers. Here, a highly versatile single‐cell assay is presented that can accommodate different cellular types, enable easy and efficient single‐cell loading and culturing, and be suitable for the study of effects of in vitro environmental factors in combination with drug screening. One salient feature of the assay is the noninvasive collection and surveying of single‐cell secretions at different time points, producing unprecedented insight of single‐cell behaviors based on the biomarker signals from individual cells under given perturbations. Above all, the acquired information is quantitative, for example, measured by the number of exosomes each single‐cell secretes for a given time period. Therefore, our single‐cell assay provides a convenient, low‐cost, and enabling tool for quantitative, time lapsed studies of single‐cell properties.     

Bone-like matrix formation on magnesium and magnesium alloys

Mg metal and its alloys have promise as a biocompatible, degradable biomaterials. This work evaluates the potential of in vitro cell culture work with osteoblast-like cells on Mg based materials, and investigates cell differentiation and growth on Mg alloyed with various non-toxic or low-toxicity elements. Mg based substrates support the adhesion, differentiation and growth of stromal cells towards an osteoblast-like phenotype with the subsequent production of a bone like matrix under in vitro conditions. No significant difference in the final tissue layer is observed on pure Mg, an AZ21 alloy or a 0.5 wt% Ca alloy. Only a 0.8 wt% Ca alloy which shows complete structural disintegration shows minimal cell growth. Due to association of non-soluble degradation products formed when Mg is incubated in physiological-like fluid, mass changes typically used to report Mg degradation are not viable estimates of degradation. Methods quantifying the time dependent change in the mechanical integrity of samples as a function of incubation time are required for a proper assessment of Mg degradation. We conclude that in vitro cell culture of bone cells on Mg substrates is expected to be a viable screening technique to assess the relative biological activity of Mg-based materials.     

Nanomolar Synthesis in Droplet Microarrays with UV‐Triggered On‐Chip Cell Screening

Miniaturization and parallelization of combinatorial organic synthesis is important to accelerate the process of drug discovery while reducing the consumption of reagents and solvents. This work presents a miniaturized platform for on‐chip solid‐phase combinatorial library synthesis with UV‐triggered on‐chip cell screening. The platform is based on a nanoporous polymer coating on a glass slide, which is modified via photolithography to yield arrays of hydrophilic (HL) spots surrounded by superhydrophobic (SH) surface. The combination of HL spots and SH background enables confinement of nanoliter droplets, functioning as miniaturized reactors for the solid‐phase synthesis. The polymer serves as support for nanomolar solid‐phase synthesis, while a photocleavable linker enables the release of the synthesized compounds into the droplets containing live cells. A 588 compound library of bisamides is synthesized via a four‐component Ugi reaction on the chip and products are detected via stamping of the droplet array onto a conductive substrate and subsequent matrix‐assisted laser desorption ionization mass spectrometry. The light‐induced cleavage shows high flexibility in screening conditions by spatial, temporal, and quantitative control.     

Real‐Time Quantification of Cell Internalization Kinetics by Functionalized Bioluminescent Nanoprobes

Cells transport mass dynamically, crossing cell membranes to maintain metabolism and systemic homeostasis, through which biomolecules are also delivered to cells for gene editing, cell reprograming, therapy, and other purposes. Quantifying the translocation kinetics is fundamentally and clinically essential, but remains limited by fluorescence‐based technologies, which are semi‐quantitative and only provide kinetics information at cellular level or in discrete time. Herein, a real‐time method of quantifying cell internalization kinetics is reported using functionalized firefly‐luciferase nanocapsules as the probe. This quantitative assay will facilitate the rational design of delivery vectors and enable high‐throughput screening of peptides and other functional molecules, constituting an effective tool for broad applications, including drug development and cancer therapy.     

A high-throughput, low-volume enzyme assay on solid support

A high-throughput enzyme assay is described that uses 1 microL or less of enzyme solution for each test. Enzyme solutions are deposited by robotic handling in a throughput of over 1000 tests/h on the surface of silica gel plates that have been preimpregnated with fluorogenic substrates. The reaction is quantitated by fluorescence. The method is compatible with water-insoluble substrates (lipases), water-soluble substrates (glycosidases), whole-protein substrates (proteases), and enzyme inhibition measurements. Hydrolytically labile umbelliferyl esters can be used to assay lipases in this format without background hydrolysis. High throughput and reproducibility were tested by fingerprint analysis of lipases and esterases against 37 different fluorogenic ester substrates. A set of eight fluorogenic unbelliferyl esters was selected for optimal activity screening of lipases and esterases on silica gel plates.     

Application of time-of-flight-secondary ion mass spectrometry for the detection of enzyme activity on solid wood substrates

Time-of-flight-secondary ion mass spectrometry (TOF-SIMS) is a surface analysis technique that is herein demonstrated to be a viable tool for the detection of enzyme activity on solid substrates. Proof-of-principle experiments are presented that utilize commercial cellulase and laccase enzymes, which are known to modify major polymeric components of wood (i.e., cellulose and lignin, respectively). Enzyme activity is assessed through principle component analysis (PCA) as well as through peak ratios intended to measure selective enzymatic wood degradation. Spectral reproducibility of the complex wood substrates is found to be within 5% relative standard deviation (RSD), allowing for relative quantification of changes in wood composition. Procedures are also presented to identify and avoid the influence of mass interferences from protein adsorption by the enzyme solutions. The activity of a cellulase cocktail is clearly evident through the TOF-SIMS spectra and is supported by high-pressure liquid chromatography (HPLC) measurements of sugar release and by complementary X-ray photoelectron spectroscopy (XPS) measurements of the wood surfaces. Laccase activity, which is mediated through small organic molecules, can be detected in the TOF-SIMS spectra through a decrease in G and S lignin peaks. This work has positive implications for the development of qualitative, high-throughput screening assays for enzyme activity on industrially relevant, lignocellulosic substrates.     

Enzyme inhibitor screening by capillary electrophoresis with an on-column immobilized enzyme microreactor created by an ionic binding technique

A novel strategy for screening the enzyme inhibitors from the complex mixtures by capillary electrophoresis with an on-column immobilized enzyme microreactor created by an ionic binding technique is reported. The enzyme microreactor was prepared in two steps: First, the capillary wall was dynamically coated with a polycationic electrolyte hexadimethrine bromide (HDB) by simply flushing the column using the HDB solution. Subsequently, a plug of the enzyme solution was injected and incubated for 5 min to permit the enzyme molecules to immobilize on the positively charged coating via ionic binding. To demonstrate this strategy, angiotensin-converting enzyme (ACE) was employed as a model for the enzyme immobilization, inhibition study, and inhibitor screening. It has been proved that such a prepared immobilized ACE microreactor displays a high enough activity and stability. Furthermore, the immobilized enzyme microreactor could be easily renewed. The inhibition study or inhibitor screening was accomplished through the following procedure: (i) the substrate solution was injected and incubated within the microreactor for a short time span; (ii) subsequently, the voltage was applied to separate the product of the enzyme reaction from the unreacted substrate based on their different mobilities, the peak area of the product representing the enzyme activity; (iii) a certain amount of enzyme inhibitor or candidate compound was spiked into the substrate solution to assay the reduction of the immobilized enzyme activity. Thus, the inhibitors can be easily identified if the reduced peak area of the product is observed in electropherograms. Because the injection volume of the capillary was only 9.8 nL and the enzyme could be reusable, the assay cost could be dramatically reduced. The screening of a small compound library containing natural extracts and commercially available inhibitors was performed. The present approach has proved to be simple, rapid, and robust.     

Homogeneous single-label biochemical Ras activation assay using time-resolved luminescence

Mutations of the small GTP-binding protein Ras have been commonly found in tumors, and Ras oncogenes have been established to be involved in the early steps of cancerogenesis. The detection of Ras activity is critical in the determination of the cell signaling events controlling cell growth and differentiation. Therefore, development of improved methods for primary screening of novel potential drugs that target small GTPase or their regulators and their signaling pathways is important. Several assays have been developed for small GTPases studies, but all these methods have limitations for a high-throughput screening (HTS) use. Multiple steps including separation, use of radioactive labels or time-consuming immunoblotting, and a need of large quantities of purified proteins are decreasing the user-friendliness of these methods. Here, we have developed a homogeneous H-Ras activity assay based on a single-label utilizing the homogeneous quenching resonance energy transfer technique (QRET). In the QRET method, the binding of a terbium-labeled GTP (Tb-GTP) to small GTPase protein H-Ras protects the signal of the label from quenching, whereas the signal of the nonbound fraction of Tb-GTP is quenched by a soluble quencher. This enables a rapid determination of the changes in the activity status of Ras. The assay optimization showed that only 60 nM concentration of purified H-Ras protein was needed. The functionality of the assay was proved by detecting the effect of H-Ras guanine nucleotide exchange factor, Son of Sevenless. The signal-to-background ratio up to 7.7 was achieved with an average assay coefficient of variation of 9.1%. The use of a low concentration of purified protein is desirable and the signal-to-background ratio of 3.4 was achieved in the assay at a concentration of 60 nM for H-Ras and SOS proteins. The need of only one labeled molecule and the ability to decrease the quantities of purified proteins used in the experiments are valuable qualities in HTS showing the potential of the QRET method.     

Investigation of enzyme kinetics using quench-flow techniques with MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is combined off-line with rapid chemical quench-flow methods to investigate the pre-steady-state kinetics of a protein-tyrosine phosphatase (PTPase). PTPase kinetics are generally interrogated spectrophotometrically by the employment of an artificial, chromophoric substrate. However, that methodology places a constraint on the experiment, hampering studies of natural, biochemically relevant substrates that do not incorporate a chromophore. The mass spectrometric assay reported herein is based on the formation of a covalent phosphoenzyme intermediate during substrate turnover. This species is generated in the reaction regardless of the substrate studied and has a molecular weight 80 Da greater than that of the native enzyme. By following the appearance of this intermediate in a time-resolved manner, we can successfully measure pre-steady-state kinetics, regardless of the incorporation of a chromophore. The strengths of the mass-spectrometric assay are its uniform response to all substrates, simple and direct detection of covalent enzyme-substrate intermediates, and facile identification of enzyme heterogeneities that may affect enzymatic activity.     

Facile One Step Formation and Screening of Tumor Spheroids Using Droplet‐Microarray Platform

Tumor spheroids or microtumors are important 3D in vitro tumor models that closely resemble a tumor's in vivo “microenvironment” compared to 2D cell culture. Microtumors are widely applied in the fields of fundamental cancer research, drug discovery, and precision medicine. In precision medicine tumor spheroids derived from patient tumor cells represent a promising system for drug sensitivity and resistance testing. Established and commonly used platforms for routine screenings of cell spheroids, based on microtiter plates of 96‐ and 384‐well formats, require relatively large numbers of cells and compounds, and often lead to the formation of multiple spheroids per well. In this study, an application of the Droplet Microarray platform, based on hydrophilic–superhydrophobic patterning, in combination with the method of hanging droplet, is demonstrated for the formation of highly miniaturized single‐spheroid‐microarrays. Formation of spheroids from several commonly used cancer cell lines in 100 nL droplets starting with as few as 150 cells per spheroid within 24–48 h is demonstrated. Established methodology carries a potential to be adopted for routine workflows of high‐throughput compound screening in 3D cancer spheroids or microtumors, which is crucial for the fields of fundamental cancer research, drug discovery, and precision medicine.     

A high-throughput HERG potassium channel function assay: an old assay with a new look

In this paper, we describe an assay using radioactive rubidium (⁸⁶Rb) efflux to screen functional human ether-a go-go-related gene (HERG) K ⁺ channels in a high-throughput screening (HTS) format. This assay offers an alternative way to examine functional interactions between chemical compounds and HERG K ⁺ channels. Follow-up experiments and discussions were carried out to address a variety of factors that affect potency evaluation within the Rb efflux assay. Factors that can affect the assay results, such as assay time, efflux rate, and compound blocking kinetics, are discussed in detail. Our results provide some explanations for the variances of the assay results and offer some guidelines for using the Rb efflux assay to evaluate compound interactions with HERG K ⁺ channels in the pharmaceutical industry.     

Spatiotemporally Controllable Peptide‐Based Nanoassembly in Single Living Cells for a Biological Self‐Portrait

Simultaneous precise localization and activity evaluation of a biomolecule in a single living cell is through an enzyme‐specific signal‐amplification process, which involves the localized, site‐specific self‐assembly, and activation of a presignaling molecule. The inactive presignaling tetraphenylethylene (TPE)‐peptide derivative, TPE‐YpYY, is nondetectable and highly biocompatible and these small molecules rapidly diffuse into living cells. Upon safely arriving at an active site, and accessing the catalytic pocket of an enzyme, TPE‐YpYY immediately and quantitatively accumulates in situ in response to enzymatic activity, forms an enzyme anchor TPE‐YYY nanoassembly, displays aggregation‐induced emission behavior, and finally lights up the active enzyme, indicating its activity, and allowing its status in living cells to be tracked. This simple and direct self‐portrait method can be used to monitor dynamic self‐assembly processes in individual living cells and may provide new insights that reveal undiscovered biological processes and that aid in developing biomedical hybrid devices. In the future, this strategy of molecular design can be further expanded to the noninvasive investigation of other bioactive molecules, thus facilitating quantitative imaging.