Microwell Array Research Articles

Controlled Rehydration of Dried Reagents for Robust Multiplex Digital PCR.

Digital polymerase chain reaction (dPCR) is emerging as a powerful method for nucleic acid detection due to its unprecedented sensitivity and precision. However, most current dPCR platforms are inherently limited by their low multiplexing ability due to primer-pair cross interactions and spectral overlap of available fluorophores. Here, we present a novel and robust method for multiplexing dPCR that is free from primer dimerization and fluorescence channel number limitation, enabling highly precise and multiplexed detection of nucleic acid targets. By prestoring target-specific primers and probes in different storage chambers, the method physically separates reactions and thus avoids the primer-pair cross interactions and spectral overlap of different fluorescent probes that usually occur within a single-tube reaction. Furthermore, a dissolvable delay valve (DDV) is embedded between each pair of the reagent prestorage chamber and reaction microwell array. Such a DDV configuration allows full reconstitution of the prestored reagents and then generates a uniform concentration distribution of the reconstituted reagents across the entire reaction microwell array, which is favorable for achieving reliable and robust multiplex dPCR assays. We demonstrated the feasibility of this method by performing an eight-plex dPCR assay targeting the seven most common point mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) and a reference sequence (wild-type KRAS allele).

Whole lifecycle observation of single-spore germinated Streptomyces using a nanogap-stabilized microfluidic chip.

Streptomyces is a model bacterium to study multicellular differentiation and the major reservoir for antibiotics discovery. However, the cellular-level lifecycle of Streptomyces has not been well studied due to its complexity and lack of research tools that can mimic their natural conditions. In this study, we developed a simple microfluidic chip for the cultivation and observation of theentire lifecycle of Streptomyces development from the single-cell perspective. The chip consists of channels for loading samples and supplying nutrients, microwell arrays for the seeding and growth of single spores, and air chambers beside the microwells that facilitate the development of aerial hyphae and spores. A unique feature of this chip is that each microwell is surrounded by a 1.5 µm nanogap connected to an air chamber, which provides a stabilized water-air interface. We used this chip to observe the lifecycledevelopment of Streptomyces coelicolor and Streptomyces griseus germinated from single spores, which revealed differentiation of aerial hyphae with progeny spores at micron-scale water-air interfaces and air chambers. Finally, we demonstrated the applicability of this chip in phenotypic assays by showing that the microbial hormone A-Factor is involved in the regulatory pathways of aerial hyphae and spore formation. The microfluidic chip could become a robust tool for studying multicellular differentiation, single-spore heterogeneity, and secondary metabolism of single-spore germinated Streptomyces.

Scalable Formation of Highly Viable and Functional Hepatocellular Carcinoma Spheroids in an Oxygen-Permeable Microwell Device for Anti-Tumor Drug Evaluation.

For high-throughput anti-cancer drug screening, microwell arrays may serve as an effective tool to generate uniform and scalable tumor spheroids. However, microwell arrays are commonly anchored in non-oxygen-permeable culture plates, leading to limited oxygen supply for avascular spheroids. Herein, a polydimethylsiloxane (PDMS)-based oxygen-permeable microwell device is introduced for generating highly viable and functional hepatocellular carcinoma (HCC) spheroids. The PDMS sheets at the bottom of the microwell device provide a high flux of oxygen like in vivo neighboring hepatic sinusoids. Owing to the better oxygen supply, the generated HepG2 spheroids are larger in size and exhibit higher viability and proliferation with less cell apoptosis and necrosis. These spheroids also exhibit lower levels of anaerobic cellular respiration and express higher levels of liver-related functions. In anti-cancer drug testing, spheroids cultured in PDMS plates show a significantly stronger resistance against doxorubicin because of the stronger stem-cell and multidrug resistance phenotype. Moreover, higher expression of vascular endothelial growth factor-A produces a stronger angiogenesis capability of the spheroids. Overall, compared to the spheroids cultured in conventional non-oxygen-permeable plates, these spheroids can be used as a more favorable model for early-stage HCCs and be applied in high-throughput anti-cancer drug screening.

High-Efficiency Generation, Drug and Radiosensitivity Test of Multicellular Tumor Spheroids by a Novel Microdevice

Compared with traditional two-dimensional culture, a three-dimensional (3D) culture platform can not only provide more reliable prediction results, but also provide a simple, inexpensive and less time-consuming method compared with animal models. A direct in vitro model of the patient’s tumor can help to achieve individualized and precise treatment. However, the existing 3D culture system based on microwell arrays has disadvantages, such as poor controllability, an uneven spheroid size, a long spheroid formation time, low-throughput and complicated operation, resulting in the need for considerable labor, etc. Here, we developed a new type of microdevice based on a 384-well plate/96-well plate microarray design. With our design, cells can quickly aggregate into clusters to form cell spheroids with better roundness. This design has the advantage of high throughput; the throughput is 33 times that of a 384-well plate. This novel microdevice is simple to process and convenient to detect without transferring the cell spheroid. The results show that the new microdevice can aggregate cells into spheroids within 24 h and can support drug and radiation sensitivity analyses in situ in approximately one week. In summary, our microdevices are fast, efficient, high-throughput, simple to process and easy to detect, providing a feasible tool for the clinical validation of individualized drug/radiation responses in patients.

Single Escherichia coli bacteria detection using a chemiluminescence digital microwell array chip

Rapid and sensitive Escherichia coli (E. coli) detection is important in determining environmental contamination, food contamination, as well as bacterial infection. Conventional methods based on bacterial culture suffer from long testing time (24 h), whereas novel nucleic acid-based and immunolabelling approaches are hindered by complicated operation, the need of complex and costly equipment, and the lack of differentiation of live and dead bacteria. Herein, we propose a chemiluminescence digital microwell array chip based on the hydrolysis of 6-Chloro-4-methylumbelliferyl-β-D-glucuronide by the β-D-glucuronidase in E. coli to achieve fast single bacterial fluorescence detection. Taking the advantage of the picoliter microwells, single bacteria are digitally encapsulated in these microwells, thus the accurate quantification of E. coli can be realized by counting the number of positive microwells. We also show that the chemiluminescence digital microwell array chip is not affected by the turbidity of the test samples as well as the temperature. Most importantly, our method can differentiate live and dead bacteria through bacterial proliferation and enzyme expression, which is confirmed by detecting E. coli after pH and chlorination treatment. By comparing with the standard method of plate counting, our method has comparable performance but significantly reduces the testing time from over 24 h–2 h and 4 h for qualitative and quantitative analysis, respectively. In addition, the microfluidic chip is portable and easy to operate without external pump, which is promising as a rapid and on-site platform for single E. coli analysis in water and food monitoring, as well as infection diagnosis.

Human Mesenchymal Stromal Cells Do Not Cause Radioprotection of Head-and-Neck Squamous Cell Carcinoma.

Radiotherapy of head-and-neck squamous cell carcinoma (HNSCC) can cause considerable normal tissue injuries, and mesenchymal stromal cells (MSCs) have been shown to aid regeneration of irradiation-damaged normal tissues. However, utilization of MSC-based treatments for HNSCC patients undergoing radiotherapy is hampered by concerns regarding potential radioprotective effects. We therefore investigated the influence of MSCs on the radiosensitivity of HNSCCs. Several human papillomavirus (HPV)-negative and HPV-positive HNSCCs were co-cultured with human bone marrow-derived MSCs using two-dimensional and three-dimensional assays. Clonogenic survival, proliferation, and viability of HNSCCs after radiotherapy were assessed depending on MSC co-culture. Flow cytometry analyses were conducted to examine the influence of MSCs on irradiation-induced cell cycle distribution and apoptosis induction in HNSCCs. Immunofluorescence stainings of γH2AX were conducted to determine the levels of residual irradiation-induced DNA double-strand breaks. Levels of connective tissue growth factor (CTGF), a multifunctional pro-tumorigenic cytokine, were analyzed using enzyme-linked immunosorbent assays. Neither direct MSC co-culture nor MSC-conditioned medium exerted radioprotective effects on HNSCCs as determined by clonogenic survival, proliferation, and viability assays. Consistently, three-dimensional microwell arrays revealed no radioprotective effects of MSCs. Irradiation resulted in a G2/M arrest of HNSCCs at 96 h independently of MSC co-culture. HNSCCs’ apoptosis rates were increased by irradiation irrespective of MSCs. Numbers of residual γH2AX foci after irradiation with 2 or 8 Gy were comparable between mono- and co-cultures. MSC mono-cultures and HNSCC-MSC co-cultures exhibited comparable CTGF levels. We did not detect radioprotective effects of human MSCs on HNSCCs. Our results suggest that the usage of MSC-based therapies for radiotherapy-related toxicities in HNSCC patients may be safe in the context of absent radioprotection.

Human Endometrium-Derived Adventitial Cell Spheroid-Loaded Antimicrobial Microneedles for Uterine Regeneration.

Asherman's syndrome (AS) occurs as a consequence of severe damage to the endometrial basalis, usually leading to menstrual abnormalities, infertility, and recurrent miscarriage in women. Currently, human endometrium-derived adventitial cells (En-ADVs) are considered ideal seed cells with high pluripotency for regenerative medicine. However, critical issues such as noninvasive repair of tissues, targeting of native stem cells, and continuous action in the injured sites are not well resolved. Herein, En-ADV spheroid-loaded hierarchical microneedles (MN/En-ADV) for in situ intrauterine repair are developed. The flexible microneedles are fabricated with gelatin methacryloyl and lactoferrin, imparting the characteristics of rapid degradation and antimicrobial activity. Benefiting from an array of microwells on microneedles, En-ADVs can rapidly form 3D cell spheroids, which display higher potential for cell proliferation, differentiation, and migration than dissociated cells. With the application of MN/En-ADV, the repaired uteri show well-defined myometrial regeneration, angiogenesis, and an increase of endometrial receptivity in a rat AS model. Notably, embryos are able to implant in the reconstructed sites and remain viable, indicating that this system promotes the restoration of both normal morphology and reproductive function in the injured uterus. It is anticipated that multifunctional MN/En-ADV can be an ideal candidate for versatile in situ tissue regeneration.

Cell-repellent polyampholyte for conformal coating on microstructures

Repellent coatings are critical for the development of biomedical and analytical devices to prevent nonspecific protein and cell adhesion. In this study, prevelex (polyampholytes containing phosphate and amine units) was synthesized for the fine coating of microdevices for cell culture. The dip-coating of the prevelex on hydrophobic substrates altered their surfaces to be highly hydrophilic and electrically neutral. The range of prebake temperature (50–150 °C) after dip-coating was moderate and within a preferable range to treat typical materials for cell culture such as polystyrene and polydimethylsiloxane. Scanning electron microscopy revealed a conformal and ultra-thin film coating on the micro/nano structures. When compared with poly(2-hydroxyethyl methacrylate) and poly(2-methacryloyloxyethyl phosphorylcholine), prevelex exhibited better characteristics for coating on microwell array devices, thereby facilitating the formation of spheroids with uniform diameters using various cell types. Furthermore, to examine cellular functionalities, mouse embryonic epithelial and mesenchymal cells were seeded in a prevelex-coated microwell array device. The two types of cells formed hair follicle germ-like aggregates in the device. The aggregates were then transplanted to generate de novo hair follicles in nude mice. The coating material provided a robust and fine coating approach for the preparation of non-fouling surfaces for tissue engineering and biomedical applications.

Multiplexed digital ELISA in picoliter droplets based on enzyme signal amplification block and precisely decoding strategy: A universal and practical biodetection platform

Ultrasensitive multiplexed digital ELISA technologies are important in clinical applications, as one of the most sensitive methods for sub-femtomolar protein detection. Compared to microwell arrays, droplet microfluidics with high-throughput droplet generation and unlimited number of partitions are the more ideal candidate for multiplexed digital biodetection. Here, a universal and practical multiplexed droplet-based digital ELISA (ddELISA) strategy was innovatively developed, and it combined fluorescent barcodes as carriers to identify multiplexed targets with the imaging based decoded method of barcodes using short and long exposure times to improve decoding accuracy. Imaging decoding of 25-plexed 3 µm dual-color barcodes in droplets with diameters of 30 µm was achieved, demonstrating that constructed bead droplet microfluidics has a high multiplexed capability. To overcome the challenge of slow mono-enzyme catalytic rate in picoliter droplets, a horseradish peroxidase polymer (polyHRP) was introduced into 14 pL microdroplets, which shortened the detection time to 3 min. As a proof of concept, 5-plexed barcodes were chosen as microcarriers to detect five cytokines in one reaction by the established ddELISA, and LODs in the sub-fM range were achieved, resulting in an up to 3–40,000-fold improvement over multiplexed suspension chip. Thus, ddELISA we developed paves a promising way for ultrasensitive multiplexed biodetection.

A self-driven carbon-doped high-density microwell array for single cell analysis

Single cell analysis preserves the heterogeneity information of target cell population in search of rare biomarkers for disease diagnosis. Microfluidic technology facilitates single cell analysis through its high integrability with multi-functionalities, high sensitivity, precision and dynamic range for digital assays. However, demonstrated microfluidic devices for single cell analysis suffer from low throughput, the need of external instruments and complicated control system. Herein, we present a self-driven high-density microwell array for quantitative analysis of single-cell metabolic activity. 38,400-microwell array (density: 25,000/cm2) is achieved through two features: (1) Two-layered vertical design of microchannels to provide more space for microwell integration; and (2) Doping of carbon powder in microwell wall to block stray light transmission and improve signal-to-noise ratio, decreasing the interval between microwells down to 30 µm. Moreover, the chip is powered by pre-stored negative pressure without the need of external pump. Our microwell array significantly reduces the assay time from over 24–3 h in Escherichia coli quantitative analysis (6-order dynamic range). We also demonstrated the viability assay and metabolic heterogeneity of single bacteria, envisioning that the microwell array could be applied for other target cells and extended to different molecular techniques such as digital PCR.

Bridging the Gap between Digital Assays and Point-of-Care Testing: Automated, Low Cost, and Ultrasensitive Detection of Thyroid Stimulating Hormone.

Medical diagnostics is moving toward disease-related target detection at very low concentrations because of the (1) quest for early-stage diagnosis, at a point where only limited target amounts are present, (2) trend toward minimally invasive sample extraction, yielding samples containing low concentrations of target, and (3) need for straightforward sample collection, usually resulting in limited volume collected. Hence, diagnostic tools allowing ultrasensitive target detection at the point-of-care (POC) are crucial for simplified and timely diagnosis of many illnesses. Therefore, we developed an innovative, fully integrated, semi-automated, and economically viable platform based on (1) digital microfluidics (DMF), enabling automated manipulation and analysis of very low sample volumes and (2) low-cost disposable DMF chips with microwell arrays, fabricated via roll-to-roll processes and allowing digital target counting. Thyroid stimulating hormone detection was chosen as a relevant application to show the potential of the system. The assay buffer was selected using design of experiments, and the assay was optimized in terms of reagent concentration and incubation time toward maximum sensitivity. The hydrophobic-in-hydrophobic microwells showed an unparalleled seeding efficiency of 97.6% ± 0.6%. A calculated LOD of 0.0013 μIU/mL was obtained, showing the great potential of the platform, especially taking into account the very low sample volume analyzed (1.1 μL). Although validation (in biological matrix) and industrialization (full automation) steps still need to be taken, it is clear that the combination of DMF, low-cost DMF chips, and digital analyte counting in microwell arrays enables the implementation of ultrasensitive and reliable target detection at the POC.

Simulation of drop impact on substrate with micro-wells

In this paper, we numerically investigate drop impact on a micro-well substrate to understand the phenomena of non-wettability. The simulation is carried out by solving three-dimensional incompressible Navier–Stokes equations using a density projection method and an adaptive grid refinement algorithm. A very sharp interface reconstruction algorithm, known as the moment-of-fluid method, is utilized to identify the multi-materials and multi-phases present in the computation domain. Our simulations predicted that a micro-well with a deep cavity can significantly reduce a solid–liquid contact in the event of drop impact. The results from the drop impact on the micro-well substrate are compared with results from drop impact on a flat substrate. Significant differences are observed between these two cases in terms of wetted area, spreading ratio, and kinetic energy. Our simulation shows that under the same conditions, a drop is more apt to jump from a micro-well substrate than from a flat surface, resulting in smaller wetted area and shorter contact time. Based on the simulation results, we draw a drop jumping region map. The micro-well substrate has a larger region than the flat surface substrate. Finally, we present a comparative analysis between a flat substrate and a substrate constructed with a dense array of micro-wells and, therefore, show that the array of micro-wells outperforms the smooth substrate with regard to non-wettability and drop wicking capability.

An integrated microfluidic system for multi-target biochemical analysis of a single drop of blood

Herein, we described an integrated microfluidic system for multi-target biochemical analysis of micro-volumes of blood samples. This system mainly consists of a microfluid handling module, an integrated microwell array chip, a temperature control module and a spectral detection module. A novel microfluidic plasma extraction approach was developed by coupling the filter membrane-based plasma separation technique with the microfluidic liquid-handling technique. With this approach, quantitative extraction of trace blood with volumes as low as a drop (30 μL) can be automatically realized. Multiple operations in multi-target biochemical analysis are achieved including micro-volume blood collecting, quantitative plasma extraction, plasma dilution, plasma distribution, transferring, biochemical reaction and absorption spectroscopy detection. This system was applied to the multi-target biochemical analysis of glucose, cholesterol and total protein in blood samples and could achieve the determination of the target analytes of a 30-μL blood sample within 11 min. The experimental results are consistent with those obtained by a commercial biochemical analyzer.

High-Throughput Microfluidics Platform for Intracellular Delivery and Sampling of Biomolecules from Live Cells.

Nondestructive cell membrane permeabilization systems enable the intracellular delivery of exogenous biomolecules for cell engineering tasks as well as the temporal sampling of cytosolic contents from live cells for the analysis of dynamic processes. Here, we report a microwell array format live-cell analysis device (LCAD) that can perform localized-electroporation induced membrane permeabilization, for cellular delivery or sampling, and directly interfaces with surface-based biosensors for analyzing the extracted contents. We demonstrate the capabilities of the LCAD via an automated high-throughput workflow for multimodal analysis of live-cell dynamics, consisting of quantitative measurements of enzyme activity using self-assembled monolayers for MALDI mass spectrometry (SAMDI) and deep-learning enhanced imaging and analysis. By combining a fabrication protocol that enables robust assembly and operation of multilayer devices with embedded gold electrodes and an automated imaging workflow, we successfully deliver functional molecules (plasmid and siRNA) into live cells at multiple time-points and track their effect on gene expression and cell morphology temporally. Furthermore, we report sampling performance enhancements, achieving saturation levels of protein tyrosine phosphatase activity measured from as few as 60 cells, and demonstrate control over the amount of sampled contents by optimization of electroporation parameters using a lumped model. Lastly, we investigate the implications of cell morphology on electroporation-induced sampling of fluorescent molecules using a deep-learning enhanced image analysis workflow.

Deep Learning‐Assisted Automated Single Cell Electroporation Platform for Effective Genetic Manipulation of Hard‐to‐Transfect Cells (Small 20/2022)

Genetic Manipulation In article number 2107795, Horacio D. Espinosa and co-workers present an automated nanopipette-based single cell electroporation system using arrays of microwells, a deep convolutional neural network for cell nuclei localization, and hardware and software for cell–nanopipette contact detection. The system enables CRISPR/Cas9 gene editing and cell perturbation studies with unprecedented throughput, efficiency, and cell viability. Applications include generation of isogenic cell lines, delivery of plasmids for reporter expression, and Cas9 ribonucleoprotein complexes for gene knockout.

Oil-Triggered and Template-Confined Dewetting for Facile and Low-Loss Sample Digitization.

This paper proposes a simple and robust method for spontaneously digitizing aqueous samples into a high-density microwell array. The method is based on an oil-triggered template-confined dewetting phenomenon. To realize the dewetting-induced sample digitization, an aqueous sample is first infused into a networked microwell array (NMA) through a pre-degassing-based self-pumping mechanism, and an immiscible oil phase is then applied over the surface of NMA chip to induce the templated dewetting. Due to periodic interfacial tension heterogeneity, such dewetting ruptures the sample at the thinnest parts (i.e., connection channels) and spontaneously splits the sample into droplets in individual microwells. Without requiring any complex pumping or valving systems, this method can discretize a sample into tens of thousands of addressable droplets in a matter of minutes with nearly 98% usage. To demonstrate the utility and universality of this self-digitization method, we exploited it to discretize samples into 40 233 wells for a digital PCR assay, the digital quantification of bacteria, the self-assembly of spherical colloidal photonic crystals, and the spherical crystallization of drugs. We believe this facile technique will provide a substantial benefit to many compartmentalized assays or syntheses where it is necessary to partition samples into a large number of small individual volumes.

Microfluidic platform for studying the anti‐cancer effect of ursolic acid on tumor spheroid

At present, the probability that a new anti-tumor drug will eventually succeed in clinical trials is extremely low. In order to make up for this shortcoming, the use of a three-dimensional (3D) cell culture model for secondary screening is often necessary. Cell spheroid is the easiest 3D model tool for drug screening. In this study, the microfluidic chip with a microwell array was manufactured, which could allow the formation of tumor spheroids with uniform size and easily retrieve cell spheroids from the chip. Cell spheroids were successfully cultured for over 15 days and the survival rate was as high as 80%. Subsequently, cellular response to the ursolic acid (UA) was observed on the chip. Compared to the monolayer culture cells in vitro, the tumor spheroids showed minor levels of epithelial-mesenchymal transition fluctuation after drug treatment. The mechanism of cell spheroid resistance to UA was further verified by detecting the expression level of upstream pathway proteins. But the invasive ability of tumor spheroids was attenuated when the duration of action of UA extended. The anti-cancer effect of UA was innovatively evaluated on breast cancer by using the microfluidic device, which could provide a basis and direction for future preclinical research on UA.

Trapping of Aqueous Droplets under Surface Acoustic Wave-Driven Streaming in Oil-Filled Microwells.

Microwell arrays are ideal platforms for cell culturing, cell separation, and low-volume liquid handling. The ability to manipulate droplets in microwells could open up the opportunity for developing new biochemical assays. Here, we study the trapping of aqueous droplets in an oil-filled microwell driven by the application of nanometer amplitude vibrations called surface acoustic waves (SAW). We elucidate the dynamics of the droplet within the vortex toward the final trapping location and the physics of the trapping phenomenon using a theoretical model by considering the relevant forces. Our study revealed that the combined effect of acoustic radiation and hydrodynamic forces leads to droplet migration and trapping. We demarcate the trapping and nontrapping regimes in terms of the minimum critical input power required for the trapping of droplets of different sizes and densities. We find that the critical power varies as the square of the droplet size and is higher for a denser droplet. The effects of input power and droplet size on the trapping location and trapping time are also studied.

Rapid cloning of antigen-specific T-cell receptors by leveraging the cis activation of T cells.

It is commonly understood that T cells are activated via trans interactions between antigen-specific T-cell receptors (TCRs) and antigenic peptides presented on major histocompatibility complex (MHC) molecules on antigen-presenting cells. By analysing a large number of T cells at the single-cell level on a microwell array, we show that T-cell activation can occur via cis interactions (where TCRs on the T cell interact with the antigenic peptides presented on MHC class-I molecules on the same cell), and that such cis activation can be used to detect antigen-specific T cells and clone their TCR within 4 d. We used the detection-and-cloning system to clone a tumour-antigen-specific TCR from peripheral blood mononuclear cells of healthy donors. TCR cloning by leveraging the cis activation of T cells may facilitate the development of TCR-engineered T cells for cancer therapy.

Selective retrieval of antibody-secreting hybridomas in cell arrays based on the dielectrophoresis

A cascade of the formation of cell arrays, the discrimination of cells secreting specific molecules, and the selective retrieval of cells has been developed to harvest antibody-secreting hybridomas in heterogeneous cell populations simply and rapidly. The microwell array device consisted of three-dimensional microband electrodes by assembling both upper and lower substrates perpendicularly. Arrays of hybridomas secreting specific antibodies were prepared by aligning hybridomas in each microwell based on the attractive force of positive dielectrophoresis (p-DEP). Antibody secreted by the hybridomas in the microwells was recognized by the antigen immobilized on the microwells or the membrane surfaces of hybridomas to discriminate hybridomas with the secretion ability. Thereafter, a repulsive force of negative dielectrophoresis (n-DEP) was applied to release the target hybridomas from the microwell array. To harvest the target hybridoma, AC signals could be modulated in the n-DEP frequency region and applied to a pair of microband electrodes located above and below each microwell containing target hybridoma. Thus, the cell-based array system described in this study allowed selective retrieval of single target hybridomas by merely switching from p-DEP to n-DEP after selecting the antibody-secreting hybridomas trapped in each microwell. The development of this high-affinity device could be useful to recover hybridomas producing antibodies in large populations of cells rapidly and effectively.