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Abstract
Flexible, robust, and user-friendly screening systems with a large dynamic range are highly desired in scientific research, industrial development, and clinical diagnostics. Droplet-based microfluidic systems with gradient concentrations of chemicals have been demonstrated as promising tools to provide confined microenvironments for screening tests with small reaction volumes. However, the generation and manipulation of gradient droplets, such as droplet merging, generally require sophisticated fluidic manipulation systems, potentially limiting their application in decentralized settings. We present a gradient-droplet SlipChip (gd-SlipChip) microfluidic device that enables instrument-free gradient droplet formation and parallel manipulation. The device can establish a gradient profile by free interfacial diffusion in a continuous fluidic channel. With a simple slipping step, gradient droplets can be generated by a surface tension-driven self-partitioning process. Additional reagents can be introduced in parallel to these gradient droplets with further slipping operations to initiate screening tests of the droplets over a large concentration range. To profile the concentration in the gradient droplets, we establish a numerical simulation model and verify it with hydrogen chloride (HCl) diffusion, as tested with a dual-color pH indicator (methyl orange and aniline blue). As a proof of concept, we tested this system with a gradient concentration of nitrofurantoin for the phenotypic antimicrobial susceptibility testing (AST) of Escherichia coli. The results of our gd-SlipChip-based AST on both reference and clinical strains of E. coli can be indicated by the bacterial growth profile within 3 h and are consistent with the clinical culture-based AST.
... The SlipChip is composed of two microfluidic plates in close contact, and fluid manipulation, such as high-density droplet array generation, 29−31 high-throughput droplet paring, 32,33 multistep droplet processing, 34−36 etc., could be achieved by simply changing the relative position of the plates via slipping processes, thus laying the foundation for SlipChip-based digital immunoassays. Due to its robustness and easy-to-use characteristic, the SlipChip technology has been applied in a variety of applications such as digital PCR, 29,37,38 immunoassay, 36 antibiotic susceptibility testing (AST), 39,40 etc. Nevertheless, the conventional SlipChip technologies have only demonstrated the capability to generate droplets of pico-or nanoliter size with total droplet number less than 30,000, which is insufficient for precise digital protein analysis with single-molecule sensitivity. ...
The emergence of digital immunoassays has advanced the sensitivity of protein analysis to ultrahigh sensitivity at the attomolar level. However, the background signal generated by the premixing of immunocomplexes and fluorogenic substrates can limit the precise quantification, especially in multiplexed assays. Herein, a bead-based SlipChip (bb-SlipChip) microfluidic device capable of massively parallel two-step sample loading is presented. The background signal can be suppressed through a two-step loading mechanism. Specifically, encapsulate the beads into the microwells first and then, through a slipping process, deliver the fluorogenic substrate in parallel into 281,200 microwells of 68 fL to perform the digital immunoassay. The quantification capability is demonstrated with a duplex assay of IL-6 and IL-10, achieving a limit of detection of 5.2 and 15.3 fg/mL, which is approximately 2-3 times improved compared to a commercial Simoa system. The bb-SlipChip provides a robust and universal method for digital immunoassay and can be extended to higher multiplexed detection as well as other biomedical applications involving microbeads.
... Some microfluidic tools including iChip [86] and SlipChip [87,88] have been successfully applied to improve bacterial isolation. It is worth noting that Ma et al. conducted a genetically targeted microfluidics-based isolation and obtained a gut bacterium listed in the human microbiome project's most wanted taxa [89] (Figure 4C). ...
Microbial resources from the human gut may find use in various applications, such as empirical research on the microbiome, the development of probiotic products, and bacteriotherapy. Due to the development of “culturomics”, the number of pure bacterial cultures obtained from the human gut has significantly increased since 2012. However, there is still a considerable number of human gut microbes to be isolated and cultured. Thus, to improve the efficiency of obtaining microbial resources from the human gut, some constraints of the current methods, such as labor burden, culture condition, and microbial targetability, still need to be optimized. Here, we overview the general knowledge and recent development of culturomics for human gut microorganisms. Furthermore, we discuss the optimization of several parts of culturomics including sample collection, sample processing, isolation, and cultivation, which may improve the current strategies.
... The SlipChip device can switch the required path for fluid operation through the sliding operation of two microfluidic plates with micro holes and channels printed in close contact, without pumps, valves and precise fluid control instruments [34]. Serial dilution has been successfully demonstrated using a self-partitioning SlipChip device for phenotypic antimicrobial susceptibility [35]. In this work, gradient droplets were generated by a surface tension-driven self-partitioning process with a simple slipping step. ...
Obesity is one of the foremost public health concerns. Human pancreatic lipase (hPL), a crucial digestive enzyme responsible for the digestion of dietary lipids in humans, has been validated as an important therapeutic target for preventing and treating obesity. The serial dilution technique is commonly used to generate solutions with different concentrations and can be easily modified for drug screening. Conventional serial gradient dilution is often performed with tedious multiple manual pipetting steps, where it is difficult to precisely control fluidic volumes at low microliter levels. Herein, we presented a microfluidic SlipChip that enabled formation and manipulation of serial dilution array in an instrument-free manner. With simple slipping steps, the compound solution could be diluted to seven gradients with the dilution ratio of 1:1 and co-incubated with the enzyme (hPL)-substrate system for screening the anti-hPL potentials. To ensure complete mixing of solution and diluent during continuous dilution, we established a numerical simulation model and conducted an ink mixing experiment to determine the mixing time. Furthermore, we also demonstrated the serial dilution ability of the proposed SlipChip using standard fluorescent dye. As a proof of concept, we tested this microfluidic SlipChip using one marketed anti-obesity drug (Orlistat) and two natural products (1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG) and sciadopitysin) with anti-hPL potentials. The IC50 values of these agents were calculated as 11.69 nM, 8.22 nM and 0.80 μM, for Orlistat, PGG and sciadopitysin, respectively, which were consistent with the results obtained by conventional biochemical assay.
Antimicrobial resistance is getting serious and becoming a threat to public health worldwide. The improper and excessive use of antibiotics is responsible for this situation. The standard methods used in clinical laboratories, to diagnose bacterial infections, identify pathogens, and determine susceptibility profiles, are time-consuming and labor-intensive, leaving the empirical antimicrobial therapy as the only option for the first treatment. To prevent the situation from getting worse, evidence-based therapy should be given. The choosing of effective drugs requires powerful diagnostic tools to provide comprehensive information on infections. Recent progress in microfluidics is pushing infection diagnosis and antimicrobial susceptibility testing (AST) to be faster and easier. This review summarizes the recent development in microfluidic assays for rapid identification and AST in bacterial infections. Finally, we discuss the perspective of microfluidic-AST to develop the next-generation infection diagnosis technologies.
In microbiological research, traditional methods for bacterial screening and antibiotic susceptibility testing are resource‐intensive. Microfluidics offers an efficient alternative with rapid results and minimal sample consumption, but the demand for cost‐effective, user‐friendly platforms persists in communities and hospitals. Inspired by the Magdeburg hemispheres, the strategy adapts to local conditions, leveraging omnipresent atmospheric pressure for self‐sealing of Rotation‐SlipChip (RSC) equipped with a 3D circular Christmas tree‐like microfluidic concentration gradient generator. This innovative approach provides an accessible and adaptable platform for microbiological research and testing in diverse settings. The RSC can avoid leakage concerns during multiple concentration gradient generation, chip‐rotating, and final long‐term incubation reaction (≥24 h). Furtherly, RSC subtypes adapted to different reactions can be fabricated in less than 15 min with cost less than $1, the result can be read through designated observational windows by naked‐eye. Moreover, the RSC demonstrates its capability for evaluating bacterial biomarker activity, enabling the rapid assessment of β‐galactosidase concentration and enzyme activity within 30 min, and the limit of detection can be reduced by 10‐fold. It also rapidly determines the minimum antibiotic inhibitory concentration and antibiotic combined medications results within 4 h. Overall, these low‐cost and user‐friendly RSC make them invaluable tools in determinations at previously impractical environment.
Antibiotic susceptibility testing (AST) is a routine procedure in diagnostic laboratories to determine pathogen resistance profiles toward antibiotics. The need for fast and accurate resistance results is rapidly increasing with a global rise in pathogen antibiotic resistance over the past years. Microfluidic technologies can enable AST with lower volumes, lower cell numbers, and a reduction in the sample-to-result time compared to state-of-the-art systems. We present a protocol to perform AST on a miniaturized nanoliter chamber array platform. The chambers are filled with antibiotic compounds and oxygen-sensing nanoprobes that serve as a viability indicator. The growth of bacterial cells in the presence of different concentrations of antibiotics is monitored; living cells consume oxygen, which can be observed as an increase of a luminesce signal within the growth chambers. Here, we demonstrate the technique using a quality control Escherichia coli strain, ATCC 35218. The AST requires 20 μL of a diluted bacterial suspension (OD600 = 0.02) and provides resistance profiles about 2–3 h after the inoculation. The microfluidic method can be adapted to other aerobic pathogens and is of particular interest for slow-growing strains.
Programmable droplet microfluidics (PDM) refers to a microfluidic device that integrates the functionality of a series of droplets with distinct spatial locations in a designated temporal order. PDM streamlines the intricate workflow of complex bioassays by enabling programmable and macroscopic droplet displacements, in which the droplets serve as reservoirs for reagents, microvalves for liquid insulation, and in some cases micropumps for mass transportation. As these droplets are intangible structures, the need for expensive microfabrication procedures is eliminated. Furthermore, the parallelization of the droplet series provides flexibility in controlling the throughput of the microfluidic analysis system. This paper provides a comprehensive review of PDMs enabled by various microfluidic mechanisms, including magnetism actuation, relative liquid displacement, capillary suction and sequential microdisplacement. Additionally, important applications of PDM systems for nucleic acid detection, immunoassay, drug testing, and sample recovery are also introduced. In conclusion, PDM demonstrates its potential as a highly advantageous tool for executing intricate multistep bioassays on a microfluidic platform. These technologies have exhibited superiority over their traditional counterparts in terms of size reduction, automation, and low sample/reagent consumption.
Simultaneously adding multiple drugs and other chemical reagents to individual droplets at specific time points presents a significant challenge, particularly when dealing with tiny droplets in high-throughput screening applications. In this study, we develop a micropatterned polymer chip as a miniaturized platform for light-induced programmable drug addition in cell-based screening. This chip incorporates a porous superhydrophobic polymer film with atom transfer radical polymerization (ATRP) reactivity, facilitating the efficient grafting of azobenzene methacrylate, a photo-conformationally changeable group, onto the hydrophilic regions of polymer matrix at targeted locations and with precise densities. By employing light irradiation, the cyclodextrin-azobenzene host-guest complexes formed on the polymer chip can switch from an "associated" to a "dissociated" state, granting precise photochemical control over the supramolecular coding system and its surface patterning ability. Significantly, the exceptional spatial and temporal control offered by these chemical transitions empowers us to utilize Digital Light Processing (DLP) systems for simultaneous regulation and release of cyclodextrin-bearing drugs across numerous droplets containing suspended or adhered cells. This approach minimizes mechanical disruption while achieving precise control over the timing of addition, dosage, and integration varieties of released drugs in high-throughput screening, all programmable to meet specific requirements. This article is protected by copyright. All rights reserved.
The rise of antimicrobial resistance (AMR) is a major global public health concern, and it is urgent to develop new antimicrobial drugs and alternative therapies. There has been growing interest in the use of phage therapy as an alternative to treat AMR, and it has shown promising results in early studies and clinical trials. Phage quantification is a crucial step in the development and application of phage therapy. The traditional double-layer plaque assay requires cumbersome manual operations and typically takes up to 18 h to yield a rough phage estimation. Spectrophotometry, flow cytometry, and PCR-based methods cannot distinguish between infectious and noninfectious phages. Here, we developed a digital biosensing method for rapid bacteriophage quantification on a digital phage SlipChip (dp-SlipChip) microfluidic device containing 2304 microdroplets in 3 nL. By compartmentalizing the phages and bacteria in nanoliter droplets and analyzing the growth profile of bacteria at 3 h, the number of infectious phages can be precisely quantified. The results from the dp-SlipChip were consistent with the traditional double-layer plaque assay method and exhibited higher consistency and repeatability. The dp-SlipChip does not require a complex fluidic handling instrument to generate and manipulate droplets. This SlipChip-based digital biosensing method not only provides a promising tool for rapid phage quantification, which is important for the use of phages in clinical practice to treat antimicrobial-resistant bacteria, but can also be used as an ultrasensitive, high-specificity method to detect bacteria. Furthermore, this approach can be applied to other digital biology studies that require analysis at the single-object level.
The clinical translations of drugs and nanomedicines depend on coherent pharmaceutical research based on biologically accurate screening approaches. Since establishing the 2D in vitro cell culture method, the scientific community has improved cell‐based drug screening assays and models. Those advances result in more informative biochemical assays and the development of 3D multicellular models to describe the biological complexity better and enhance the simulation of the in vivo microenvironment. Despite the overall dominance of conventional 2D and 3D cell macroscopic culture methods, they present physicochemical and operational challenges that impair the scale‐up of drug screening by not allowing a high parallelization, multidrug combination, and high‐throughput screening. Their combination and complementarity with microfluidic platforms enable the development of microfluidics‐based cell culture platforms with unequivocal advantages in drug screening and cell therapies. Thus, this review presents an updated and consolidated view of cell culture miniaturization's physical, chemical, and operational considerations in the pharmaceutical research scenario. It clarifies advances in the field using gradient‐based microfluidics, droplet‐based microfluidics, printed‐based microfluidics, digital‐based microfluidics, SlipChip, and paper‐based microfluidics. Finally, it presents a comparative analysis of the performance of cell‐based methods in life research and development to achieve increased precision in the drug screening process.
Empirical antibiotic therapies are prescribed for treating uncomplicated urinary tract infections (UTIs) due to the long turnaround time of conventional antimicrobial susceptibility testing (AST), leading to the prevalence of multi-drug resistant pathogens. We present a ready-to-use 3D microwell array chip to directly conduct comprehensive AST of pathogenic agents in urine at the single-cell level. The developed device features a highly integrated 3D microwell array, offering a dynamic range from 102 to 107 CFU mL-1, and a capillary valve-based flow distributor for flow equidistribution in dispensing channels and uniform sample distribution. The chip with pre-loaded reagents and negative pressure inside only requires the user to initiate AST by loading samples (∼3 s) and can work independently. We demonstrate an accessible sample-to-result workflow, including syringe filter-based bacteria separation and rapid single-cell AST on chip, which enables us to bypass the time-consuming bacteria isolation and pre-culture, speeding up the AST in ∼3 h from 2 days of conventional methods. Moreover, the bacterial concentration and AST with minimum inhibitory concentrations can be assessed simultaneously to provide comprehensive information on infections. With further development for multiple antibiotic conditions, the Dsc-AST assay could contribute to timely prescription of targeted drugs for better patient outcomes and mitigation of the threat of drug-resistant bacteria.
The first step in droplet-based microfluidic systems is droplet generation. For achieving the desired performance of a droplet-based microfluidic system one needs to control the droplet generator. In the present study, we propose the microfluidic equivalents of three logical systems using a passive method with a kind of trap and release technique that can be employed to activate (set latch), deactivate (reset latch), or change the droplet production line (set-reset latch) in a droplet-based microfluidic device. The operation of such systems has been evaluated and discussed by solving the Navier–Stokes equations coupled with the level-set method.
With the prevalence of bacterial infections and increasing levels of antibiotic resistance comes the need for rapid and accurate methods for bacterial classification (BC) and antibiotic susceptibility testing (AST). Here we demonstrate the use of the fluid handling technique digital microfluidics (DMF) for automated and simultaneous BC and AST using growth metabolic markers. Custom instrumentation was developed for this application including an integrated heating module and a machine-learning-enabled low-cost colour camera for real-time absorbance and fluorescent sample monitoring on multipurpose devices. Antibiotic dilutions along with sample handling, mixing and incubation at 37 °C were all pre-programmed and processed automatically. By monitoring the metabolism of resazurin, resorufin beta-D-glucuronide and resorufin beta-D-galactopyranoside to resorufin, BC and AST were achieved in under 18 h. AST was validated in two uropathogenic E. coli strains with antibiotics ciprofloxacin and nitrofurantoin. BC was performed independently and simultaneously with ciprofloxacin AST for E. coli, K. pneumoniae, P. mirabilis and S. aureus. Finally, a proof-of-concept multiplexed system for breakpoint testing of two antibiotics, as well as E. coli and coliform classification was investigated with a multidrug-resistant E. coli strain. All bacteria were correctly identified, while AST and breakpoint test results were in essential and category agreement with reference methods. These results show the versatility and accuracy of this all-in-one microfluidic system for analysis of bacterial growth and phenotype.
Stenotrophomonas maltophilia causes high mortality infections in immunocompromised hosts with limited therapeutic options. Many U.S. laboratories rely on commercial automated antimicrobial susceptibility tests (cASTs) and use CLSI breakpoints (BPs) for S. maltophilia . However, contemporary data on these systems is lacking. We assessed performances of Vitek2, MicroScan Walkaway and Phoenix relative to reference broth microdilution for trimethoprim-sulfamethoxazole (SXT), levofloxacin (LEV), minocycline (MIN) and ceftazidime (CAZ), with 109 S. maltophilia bloodstream isolates. Using CLSI breakpoints, categorical agreement (CA) was below 90% on all systems and drugs, with the exception of SXT by MicroScan (98.1%) and Phoenix (98.1%) and MIN by MicroScan (100%) and Phoenix (99.1%). For SXT, Vitek2 yielded a 77.1% CA. LEV and CAZ CA ranged from 67% - 85%. Very major errors (VME) were >3% for SXT (MicroScan, Phoenix), LEV (MicroScan) and CAZ (all systems). Major errors (ME) were >3% for SXT (Vitek 2), LEV (Phoenix) and CAZ (MicroScan, Phoenix). Minor errors were >10% for CAZ and LEV on all systems. Data were analyzed with EUCAST pharmacokinetic/pharmacodynamic CAZ, LEV, ciprofloxacin (CIP) and tigecycline (TGC) breakpoints when possible. CA was <90% for all. VME were >3% for CAZ (all systems), LEV (MicroScan), and TGC (Vitek2) and ME were >3% for LEV (MicroScan), CAZ (all systems), ciprofloxacin (Vitek2 and MicroScan) and TGC (Vitek 2, Phoenix). Minor errors (MI) were >10% for all agents and systems, by EUCAST breakpoints with an intermediate category (LEV, CAZ, CIP). Laboratories should use caution with cASTs for S. maltophilia as a high rate of errors may be observed.
Inefficiency of medical therapies used in order to cure patients with bacterial infections requires not only to actively look for new therapeutic strategies but also to carefully select antibiotics based on variety of parameters, including microbiological. Minimal inhibitory concentration (MIC) defines in vitro levels of susceptibility or resistance of specific bacterial strains to applied antibiotic. Reliable assessment of MIC has a significant impact on the choice of a therapeutic strategy, which affects efficiency of an infection therapy. In order to obtain credible MIC, many elements must be considered, such as proper method choice, adherence to labeling rules, and competent interpretation of the results. In this paper, two methods have been discussed: dilution and gradient used for MIC estimation. Factors which affect MIC results along with the interpretation guidelines have been described. Furthermore, opportunities to utilize MIC in clinical practice, with pharmacokinetic /pharmacodynamic parameters taken into consideration, have been investigated. Due to problems related to PK determination in individual patients, statistical estimation of the possibility of achievement of the PK/PD index, based on the Monte Carlo, was discussed. In order to provide comprehensive insights, the possible limitations of MIC, which scientists are aware of, have been outlined.
Currently, there are no time‐saving and cost‐effective high‐throughput screening methods for the evaluation of bacterial drug‐resistance. In this study, a droplet microarray (DMA) system is established as a miniaturized platform for high‐throughput screening of antibacterial compounds using the emerging, opportunistic human pathogen Pseudomonas aeruginosa (P. aeruginosa) as a target. Based on the differences in wettability of DMA slides, a rapid method for generating microarrays of nanoliter‐sized droplets containing bacteria is developed. The bacterial growth in droplets is evaluated using fluorescence. The new method enables immediate screening with libraries of antibiotics. A novel simple colorimetric readout method compatible with the nanoliter size of the droplets is established. Furthermore, the drug‐resistance of P. aeruginosa 49, a multi‐resistant strain from an environmental isolate, is investigated. This study demonstrates the potential of the DMA platform for the rapid formation of microarrays of bacteria for high‐throughput drug screening.
Infectious diseases caused by multidrug resistant (MDR) bacterial pathogens are impending threats to global health. Since delays in identifying drug resistance would significantly increase mortality, fast and accurate antibiotic susceptibility tests (ASTs) are critical for addressing antibiotic resistant issue. However, the conventional methods for ASTs are always labor-intensive, imprecise, complex and slow (taking 2-3 days). To address these issues, some advanced microfluidics systems are designed for rapid phenotypic and genotypic analysis of antibiotic resistance. This review highlights the recent development of microfluidics-based ASTs at single-cell or single-molecule level for guiding antibiotic treatment decisions and predicting therapeutic outcomes.
Antimicrobial resistance (AMR) is a major threat to human health worldwide, and the rapid detection and quantification of resistance, combined with antimicrobial stewardship, are key interventions to combat the spread and emergence of AMR. Antimicrobial susceptibility testing (AST) systems are the collective set of diagnostic processes that facilitate the phenotypic and genotypic assessment of AMR and antibiotic susceptibility. Over the past 30 years, only a few high-throughput AST methods have been developed and widely implemented. By contrast, several studies have established proof of principle for various innovative AST methods, including both molecular-based and genome-based methods, which await clinical trials and regulatory review. In this Review, we discuss the current state of AST systems in the broadest technical, translational and implementation-related scope. In this Review, van Belkum and colleagues discuss routinely used antimicrobial susceptibility testing (AST) methods, explore current efforts to improve phenotypic AST systems — including new emerging technologies as well as genomic and gene-based antimicrobial resistance detection methods — and highlight the challenges and opportunities for new rapid AST systems.
Objective:
For evaluating the antibiotic resistance of Helicobacter pylori, the agar dilution method is the gold standard; however, using this method in daily practice is laborious. E-test has been proposed to be an uncomplicated method. This study was aimed at validating the E-test and detecting the presence of any bias between the agar dilution method and E-test.
Results:
The agar dilution method and E-test were performed using five antibiotics for 72 strains of H. pylori obtained from clinical patients in Indonesia. The E-test's results showed a higher prevalence of resistance to all the antibiotics tested but the difference was not significant. Results showed high essential agreement (> 90.0%) for all the antibiotics, but only 84.7% for metronidazole. The agreement for MIC value was acceptable for levofloxacin, clarithromycin, and metronidazole. For amoxicillin, it showed only fair agreement (0.25) by the Kappa analysis and significant difference by Passing-Bablok regression. Even though some discrepancies were found, the E-test has an acceptable agreement for levofloxacin, metronidazole, tetracycline, and clarithromycin but further confirmation may be necessary for amoxicillin.
Combinatorial screening is frequently used to identify chemicals with synergistic effects by measuring the response of biological entities exposed to various chemical-dose combinations. Conventional microwell-based combinatorial screening is resource-demanding, and the closed microfluidics-based screening requires sophisticated fluidic control systems. In this work, we present a novel combinatorial screening platform based on the surface energy trap (SET)-assisted magnetic digital microfluidics. This platform, known as FlipDrop, rapidly generates chemical combinations by coupling two droplet arrays with orthogonal chemical concentration gradients with a simple flip. We have illustrated the working principle of FlipDrop by generating combinations of quantum dots. We have also successfully demonstrated the screening of quantum dot fluorescence resonance energy transfer (QD-FRET) on the FlipDrop platform by measuring the FRET response. This report demonstrates that FlipDrop is capable of rapidly generating chemical combinations with unprecedented ease for combinatorial screening.
Antibiotic susceptibility testing (AST) specifies effective antibiotic dosage and formulates a profile of empirical therapy for the proper management of an individual patient’s health against deadly infections. Therefore, rapid diagnostic plays a pivotal role in the treatment of bacterial infection. In this article, the authors review the socio-economic burden and emergence of antibiotic resistance. An overview of the phenotypic, genotypic, and emerging techniques for AST has been provided and discussed, highlighting the advantages and limitations of each. The historical perspective on conventional methods that have paved the way for modern AST like disk diffusion, Epsilometer test (Etest), and microdilution, is presented. Several emerging methods, such as microfluidic-based optical and electrochemical AST have been critically evaluated. Finally, the challenges related with AST and its outlook in the future are presented.
Performing drug screening of tissue derived from cancer patient biopsies using physiologically relevant 3D tumour models presents challenges due to the limited amount of available cell material. Here, we present a microfluidic platform that enables drug screening of cancer cell-enriched multicellular spheroids derived from tumour biopsies, allowing extensive anticancer compound screening prior to treatment. This technology was validated using cell lines and then used to screen primary human prostate cancer cells, grown in 3D as a heterogeneous culture from biopsy-derived tissue. The technology enabled the formation of repeatable drug concentration gradients across an array of spheroids without external fluid actuation, delivering simultaneously a range of drug concentrations to multiple sized spheroids, as well as replicates for each concentration. As proof-of-concept screening, spheroids were generated from two patient biopsies and a panel of standard-of-care compounds for prostate cancer were tested. Brightfield and fluorescence images were analysed to provide readouts of spheroid growth and health, as well as drug efficacy over time. Overall, this technology could prove a useful tool for personalised medicine and future drug development, with the potential to provide cost- and time-reduction in the healthcare delivery.
Significance
Antibiotic resistance is a global threat to human health. The problem is aggravated by unnecessary and incorrect use of broad spectrum antibiotics. One way to provide correct treatment and slow down the development of antibiotic resistance is to assay the susceptibility profile of the infecting bacteria before treatment is initiated and let this information guide the choice of antibiotic. Here, we present an antibiotic susceptibility test that is sufficiently fast to be used at the point of care. We show that it is possible to determine if a urinary tract infection is caused by resistant bacteria within 30 min of loading a urine sample, even if the bacterial concentration in the urine is very low.
Various concentration gradient generation methods based on microfluidic systems are summarized in this paper. The review covers typical structural characteristics, gradient generation mechanisms, theoretical calculation formulas, applicable scopes, and advantages and disadvantages of these approaches in detail. According to the type of reagents involved, these methods are classified into mono-phase methods and multi-phase methods, both of which can be implemented by alternative protocols, while the latter methods particularly refer to droplet-based platforms. For mono-phase methods, the shearing effect would be presented if there are flowing streams in the gradient generation channel. Therefore, the generation speed of channels with moving liquids is relatively fast, which is suitable for dynamic gradients but accompanied by shearing as well, while channels without flowing streams would avoid shearing but are prone to static gradient generation determined by the low speed. Newly developed droplet-based generation systems could provide isolated droplets to avoid the disturbances from the outside continuous phase, however, they require precise droplet generation and control modules. Thereby the most suitable platform can be chosen according to the specific application, while the advantages of different methods could be combined to evade the defects and improve the precision of a single structure.
For personalized screening and therapeutic inventions across many diseases, the drug–dose response for an individual patient is a major unmet need. In this work, we applied a direct strategy to generate a static microwell array for cell culture with uniform cell seeding and to create the desired chemical concentration gradient into this cell array. It is a simple, novel and easily operable device for a high-throughput drug–dose response experiment that complies with the procedures of cell-based drug screening. Only two repeated operating steps suffice for the entire cell-based drug test—an injection of cells or drug into the flow channel and then injection of air flow into the flow channel. This device comprises two PDMS layers: one side forms an air chamber; the other side forms the liquid channel with embedded cavities as the cells culture and reaction region. The concentration of the drug decreases exponentially along the microwell array; the range of the concentration gradient is varied with the rate of air flow, the volume of the drug plug and the initial concentration of the drug. Small variations of concentration were accessed across varied ranges; the IC50 value of DOX in the MDA-MB-231 cell line was thus utilized on this device, precisely and quickly. The IC50 value calculated in this work is consistent with the range published elsewhere. With this device, hundreds of data points per compound drug screening can be tested in one experiment, which will be an essential key to determine customized drug dosage and to make possible personalized medicine.
Inspired by the paper platforms for 3-D cell culture, a paper-based microfluidic device containing drug concentration gradient was designed and constructed for investigating cell response to drugs based on high throughput analysis. This drug gradient generator was applied to generate concentration gradients of doxorubicin (DOX) as the model drug. HeLa cells encapsulated in collagen hydrogel were incubated in the device reservoirs to evaluate the cell viability based on the controlled release of DOX spatially. It was demonstrated that drug diffusion through the paper fibers created a gradient of drug concentration, which influenced cell viability. This drug screening platform has a great opportunity to be applied for drug discovery and diagnostic studies with simultaneous and parallel tests of drugs under various gradient concentrations.
This paper reports a polydimethylsiloxane (PDMS) SlipChip for in vitro cell culture applications, multiple-treatment assays, cell co-cultures, and cytokine detection assays. The PDMS SlipChip is composed of two PDMS layers with microfluidic channels on each surface that are separated by a thin silicone fluid (Si-fluid) layer. The integration of Si-fluid enables the two PDMS layers to be slid to different positions; therefore, the channel patterns can be re-arranged for various applications. The SlipChip design significantly reduces the complexity of sample handling, transportation, and treatment processes. To apply the developed SlipChip for cell culture applications, human lung adenocarcinoma epithelial cells (A549) and lung fibroblasts (MRC-5) were cultured to examine the biocompatibility of the developed PDMS SlipChip. Moreover, embryonic pluripotent stem cells (ES-D3) were also cultured in the device to evaluate the retention of their stemness in the device. The experimental results show that cell morphology, viability and proliferation are not affected when the cells are cultured in the SlipChip, indicating that the device is highly compatible with mammalian cell culture. In addition, the stemness of the ES-D3 cells was highly retained after they were cultured in the device, suggesting the feasibility of using the SlipChip for stem cell research. Various cell experiments, such as simultaneous triple staining of cells and co-culture of MRC-5 with A549 cells, were also performed to demonstrate the functionalities of the PDMS SlipChip. Furthermore, we used a cytokine detection assay to evaluate the effect of endotoxin (lipopolysaccharides, LPS) treatment on the cytokine secretion of A549 cells using the SlipChip. The developed PDMS SlipChip provides a straightforward and effective platform for various on-chip in vitro cell cultures and consequent analysis, which is promising for a number of cell biology studies and biomedical applications.
This paper describes a simple and reusable microfluidic SlipChip device for studying bacterial chemotaxis based on free interface diffusion. The device consists of two glass plates with reconfigurable microwells and ducts, which can set up 20 parallel chemotaxis units as duplicates. In each unit, three nanoliter microwells and connecting ducts were assembled for pipette loading of a chemoeffector solution, bacterial suspension, and 1X PBS buffer solution. By a simple slipping operation, three microwells were disconnected from other units and interconnected by the ducts, which allowed the formation of diffusion concentration gradients of the chemoeffector for inducing cell migration from the cell microwell towards the other two microwells. The migration of cells in the microwells was monitored and accurately counted to evaluate chemotaxis. Moreover, the migrated cells were easily collected by pipetting for further studies after a slip step to reconnect the chemoeffector microwells. The performance of the device was characterized by comparing chemotaxis of two Escherichia coli species, using aspartic acid as the attractant and nitrate sulfate as the repellent. It also enables the separation of bacterial species from a mixture, based on the difference of chemotactic abilities, and collection of the cells with strong chemotactic phenomena for further studies off the chip.
This account examines developments in "digital" biology and chemistry within the context of microfluidics, from a personal perspective. Using microfluidics as a frame of reference, we identify two areas of research within digital biology and chemistry that are of special interest: (i) the study of systems that switch between discrete states in response to changes in chemical concentration of signals, and (ii) the study of single biological entities such as molecules or cells. In particular, microfluidics accelerates analysis of switching systems (i.e., those that exhibit a sharp change in output over a narrow range of input) by enabling monitoring of multiple reactions in parallel over a range of concentrations of signals. Conversely, such switching systems can be used to create new kinds of microfluidic detection systems that provide "analog-to-digital" signal conversion and logic. Microfluidic compartmentalization technologies for studying and isolating single entities can be used to reconstruct and understand cellular processes, study interactions between single biological entities, and examine the intrinsic heterogeneity of populations of molecules, cells, or organisms. Furthermore, compartmentalization of single cells or molecules in "digital" microfluidic experiments can induce switching in a range of reaction systems to enable sensitive detection of cells or biomolecules, such as with digital ELISA or digital PCR. This "digitizing" offers advantages in terms of robustness, assay design, and simplicity because quantitative information can be obtained with qualitative measurements. While digital formats have been shown to improve the robustness of existing chemistries, we anticipate that in the future they will enable new chemistries to be used for quantitative measurements, and that digital biology and chemistry will continue to provide further opportunities for measuring biomolecules, understanding natural systems more deeply, and advancing molecular and cellular analysis. Microfluidics will impact digital biology and chemistry and will also benefit from them if it becomes massively distributed.
We describe the development of a fully automatic and programmable microfluidic cell culture array that integrates on-chip generation of drug concentrations and pair-wise combinations with parallel culture of cells for drug candidate screening applications. The device has 64 individually addressable cell culture chambers in which cells can be cultured and exposed either sequentially or simultaneously to 64 pair-wise concentration combinations of two drugs. For sequential exposure, a simple microfluidic diffusive mixer is used to generate different concentrations of drugs from two inputs. For generation of 64 pair-wise combinations from two drug inputs, a novel time dependent variable concentration scheme is used in conjunction with the simple diffusive mixer to generate the desired combinations without the need for complex multi-layer structures or continuous medium perfusion. The generation of drug combinations and exposure to specific cell culture chambers are controlled using a LabVIEW interface capable of automatically running a multi-day drug screening experiment. Our cell array does not require continuous perfusion for keeping cells exposed to concentration gradients, minimizing the amount of drug used per experiment, and cells cultured in the chamber are not exposed to significant shear stress continuously. The utility of this platform is demonstrated for inducing loss of viability of PC3 prostate cancer cells using combinations of either doxorubicin or mitoxantrone with TRAIL (TNF-alpha Related Apoptosis Inducing Ligand) either in a sequential or simultaneous format. Our results demonstrate that the device can capture the synergy between different sensitizer drugs and TRAIL and demonstrate the potential of the microfluidic cell array for screening and optimizing combinatorial drug treatments for cancer therapy.
Cells in their native microenvironment are subjected to varying combinations of biochemical cues and mechanical cues in a wide range. Although many signaling pathways have been found to be responsive for extracellular cues, little is known about how biochemical cues crosstalk with mechanical cues in a complex microenvironment. Here, we introduced heterogeneous droplets on a microchip, which were rapidly assembled by combining wettability-patterned microchip and programmed droplet manipulations, for a high-throughput cell screening of the varying combinations of biochemical cues and mechanical cues. This platform constructed a heterogeneous droplet/microgel array with orthogonal gradual chemicals and materials, which was further applied to analyze the cellular Wnt/β-catenin signaling in response to varying combinations of Wnt ligands and substrate stiffness. Thus, this device provides a powerful multiplexed bioassay platform for drug development, tissue engineering, and stem cell screening.
Pancreatic cancer is a leading cause of cancer death worldwide and its global burden has more than doubled over the past 25 years. The highest incidence regions for pancreatic cancer include North America, Europe and Australia, and although much of this increase is due to ageing worldwide populations, there are key modifiable risk factors for pancreatic cancer such as cigarette smoking, obesity, diabetes and alcohol intake. The prevalence of these risk factors is increasing in many global regions, resulting in increasing age-adjusted incidence rates for pancreatic cancer, but the relative contribution from these risk factors varies globally due to variation in the underlying prevalence and prevention strategies. Inherited genetic factors, although not directly modifiable, are an important component of pancreatic cancer risk, and include pathogenic variants in hereditary cancer genes, genes associated with hereditary pancreatitis, as well as common variants identified in genome-wide association studies. Identification of the genetic changes that underlie pancreatic cancer not only provides insight into the aetiology of this cancer but also provides an opportunity to guide early detection strategies. The goal of this Review is to provide an up-to-date overview of the established modifiable and inherited risk factors for pancreatic cancer.
Antimicrobial resistance is a growing problem, necessitating rapid antimicrobial susceptibility testing (AST) to enable effective in-clinic diagnostic testing and treatment. Conventional AST using broth microdilution or the Kirby-Bauer disk diffusion are time-consuming (e.g., 24-72 h), labor-intensive, and costly and consume reagents. Here, we propose a novel gradient-based microchamber microfluidic (GM2) platform to perform AST assay for a wide range of antibiotic concentrations plus zero (positive control) and maximum (negative control) concentrations all in a single test. Antibiotic lateral diffusion within enriched to depleted (Cmax and zero, respectively) cocurrent flowing fluids, moving alongside a micron-sized main channel, is led to form an antibiotic concentration profile in microchambers, connected to the depleted side of the main channel. We examined the tunability of the GM2 platform, in terms of producing a wide range of antibiotic concentrations in a gradient mode between two consecutive microchambers with changing either the loading fluids' flow rates or their initial concentrations. We also tested the GM2 platform for profiling bacteria associated with human Crohn's disease and bovine mastitis. Time to result for performing a complete AST assay was ∼ 3-4 h in the GM2 platform. Lastly, the GM2 platform tracked the bacterial growth independent of an antibiotic mechanism of action or bacterial species in a robust and easy-to-implement fashion.
This paper presents a portable integrated digital PCR (PI-dPCR) system with a self-partitioning SlipChip (sp-SlipChip) microfluidic device for the quantitative analysis of BK virus (BKV) viral load directly from raw urine samples. Digital PCR is an accurate nucleic acid quantification method with single-molecule sensitivity, but the complexity of the instrument and the limited integration of the operation workflow greatly limit its application in clinical diagnostics, especially point-of-care testing (PoCT). Our PI-dPCR system has a small footprint, is lightweight, and is fully integrated with the thermal control and fluorescence imaging modules. Unlike the traditional SlipChip device, which requires the precise overlapping of microfeatures on the contacting surfaces to establish the fluidic loading path, this sp-SlipChip device utilizes microchannels with alternating depth and width for fluidic manipulation. This system can quantify BKV directly from raw urine samples with a simple “sample-to-digital-result” operation workflow without complex nucleic acid extraction and purification steps. The current design of the system provides a dynamic range of 3.0 × 10⁴ to 1.5 × 10⁸ copies/mL of BKV DNA in clinical urine samples within 2 h. We tested the system for the quantification of BKV viral load in thirty archived urine samples from kidney transplantation recipients and twelve additional samples from six patients before and after the adjustment of immunosuppressive treatment. This integrated system provides a promising method for both the detection and monitoring of viral infection in a point-of-care setting.
To accelerate the discovery of anti-cancer drugs, there is an urgent need to establish an inexpensive and simple preclinical model that can simulate the tumor microenvironment and screen the drug candidates. Some widely used screening platforms are high-cost and over-simple to verify the cell-drug interactions for predicting human clinical trial outcomes. As a promising technology, microfluidic chip can replace traditional screening methods due to low reagent consumption and the capability of recapitulating the 3D cell culture more representative of the native tumor microenvironment. This review covers the materials for microchip fabrication (especially hydrogel), tumor microenvironment, 2D and 3D tumor culture on chips, drug screening methods (concentration gradient generator-based, microdroplet-based, microarray-based chips, and tumor tissue chip), and their pros and cons for cancer treatment. We also discuss the future development of microfluidic for anti-cancer drug discovery research.
UTIs are amongst the most frequent bacterial infections. However, the clinical phenotypes of UTI are heterogeneous and range from rather benign, uncomplicated infections to complicated UTIs (cUTIs), pyelonephritis and severe urosepsis. Stratification of patients with UTIs is, therefore, important. Several classification systems exist for the description and classification of UTIs, with the common rationale that cUTIs have a higher risk of recurrence or chronification, progression or severe outcome than uncomplicated UTIs. The pathophysiology and treatment of cUTIs and pyelonephritis are driven more by host factors than by pathogen attributes. cUTIs and pyelonephritis are associated with high antimicrobial resistance rates among causative pathogens. However, antimicrobial resistance rates can differ substantially, depending on the population being studied and whether the data being analysed are from surveillance studies, registry data or interventional studies, in which specific inclusion and exclusion criteria are used for patient selection. For example, antibiotic resistance rates are higher in patients with urosepsis than in those with less severe infections. Thus, treatment outcomes differ substantially among studies, ranging from 50% to almost 100% clearance of infection, depending on the patient population analysed, the UTI entities included and the primary outcome of the study. Pyelonephritis and cUTIs have emerged as infection models for the study of novel antibiotics, including extensive investigation of novel substances active against Gram-negative bacteria.
Spatiotemporal manipulation of extracellular chemical environments with simultaneous monitoring of cellular responses plays an essential role in exploring fundamental biological processes and expands our understanding of underlying mechanisms. Despite the rapid progress and promising successes in manipulation strategies, many challenges remain due to the small size of cells and the rapid diffusion of chemical molecules. Fortunately, emerging microfluidic technology has become a powerful approach for precisely controlling the extracellular chemical microenvironment, which benefits from its integration capacity, automation, and high-throughput capability, as well as its high resolution down to submicron. Here, we summarize recent advances in microfluidics manipulation of the extracellular chemical microenvironment, including the following aspects: i) Spatial manipulation of chemical microenvironments realized by convection flow-, diffusion-, and droplet-based microfluidics, and surface chemical modification; ii) Temporal manipulation of chemical microenvironments enabled by flow switching/shifting, moving/flowing cells across laminar flows, integrated microvalves/pumps, and droplet manipulation; iii) Spatiotemporal manipulation of chemical microenvironments implemented by a coupling strategy and open-space microfluidics; and iv) High-throughput manipulation of chemical microenvironments. Finally, we briefly present typical applications of the above-mentioned technical advances in cell-based analyses including cell migration, cell signaling, cell differentiation, multicellular analysis, and drug screening. We further discuss the future improvement of microfluidics manipulation of extracellular chemical microenvironments to fulfill the needs of biological and biomedical research and applications.
Human papillomavirus (HPV) is one of the most common sexually transmitted infections worldwide, and persistent HPV infection can cause warts and even cancer. Nucleic acid analysis of HPV viral DNA can be very informative for the diagnosis and monitoring of HPV. Digital nucleic acid analysis, such as digital PCR and digital isothermal amplification, can provide sensitive detection and precise quantification of target nucleic acids, and its utility has been demonstrated in many biological research and medical diagnostic applications. A variety of methods have been developed for the generation of a large number of individual reaction partitions, a key requirement for digital nucleic acid analysis. However, an easily assembled and operated device for robust droplet formation without preprocessing devices, auxiliary instrumentation or control systems is still highly desired. In this paper, we present a self-partitioning SlipChip (sp-SlipChip) microfluidic device for the slip-induced generation of droplets to perform digital loop-mediated isothermal amplification (LAMP) for the detection and quantification of HPV DNA. In contrast to traditional SlipChip methods, which require the precise alignment of microfeatures, this sp-SlipChip utilized a design of “chain-of-pearls” continuous microfluidic channel that is independent of the overlapping of microfeatures on different plates to establish the fluidic path for reagent loading. Initiated by a simple slipping step, the aqueous solution can robustly self-partition into individual droplets by capillary pressure-driven flow. This advantage makes the sp-SlipChip very appealing for the point-of-care quantitative analysis of viral load. As a proof of concept, we performed digital LAMP on an sp-SlipChip to quantify human papillomaviruses (HPVs) 16 and 18 and tested this method with fifteen anonymous clinical samples.
With ever increasing drug resistance and emergence of new diseases, demand for new drug development is at an unprecedented urgency. This fact has led to extensive recent efforts to develop new drugs and novel techniques for efficient drug screening. However, new drug development is commonly hindered by cost and time span. Thus, developing more accessible, cost-effective methods for drug screening is necessary. Compared with conventional drug screening methods, a microfluidic-based system has superior advantages in sample consumption, reaction time, and cost of the operation. In this paper, the advantages of microfluidic technology in drug screening as well as the critical factors for device design are described. The strategies and applications of microfluidics for drug screening are reviewed. Moreover, current limitations and future prospects for a drug screening microdevice are also discussed.
Superbugs such as infectious bacteria pose a great threat to humanity due to an increase in bacterial mortality leading to clinical treatment failure, lengthy hospital stay, intravenous therapy and accretion of bacteraemia. These disease-causing bacteria gain resistance to drugs over time which further complicates the treatment. Monitoring of antibiotic resistance is therefore necessary so that bacterial infectious diseases can be diagnosed rapidly. Antimicrobial susceptibility testing (AST) provides valuable information on the efficacy of antibiotic agents and their dosages for treatment against bacterial infections. In clinical laboratories, most widely used AST methods are disk diffusion, gradient diffusion, broth dilution, or commercially available semi-automated systems. Though these methods are cost-effective and accurate, they are time-consuming, labour-intensive, and require skilled manpower. Recently much attention has been on developing rapid AST techniques to avoid misuse of antibiotics and provide effective treatment. In this review, we have discussed emerging engineering AST techniques with special emphasis on phenotypic AST. These techniques include fluorescence imaging along with computational image processing, surface plasmon resonance, Raman spectra, and laser tweezer as well as micro/nanotechnology-based device such as microfluidics, microdroplets, and microchamber. The mechanical and electrical behaviour of single bacterial cell and bacterial suspension for the study of AST is also discussed.
Serial dilution is a commonly used technique that generates a low-concentration working sample from a high-concentration stock solution and is used to set up screening conditions over a large dynamic range for biological study, optimization of reaction conditions, drug screening, etc. Creating an array of serial dilutions usually requires cumbersome manual pipetting steps or a robotic liquid handling system. Moreover, it is very challenging to set up an array of serial dilutions in nanoliter volumes in miniaturized assays. Here, a multistep SlipChip microfluidic device is presented for generating serial dilution nanoliter arrays in high throughput with a series of simple sliding motions. The dilution ratio can be precisely predetermined by the volumes of mother microwells and daughter microwells, and this paper demonstrates devices designed to have dilution ratios of 1:1, 1:2, and 1:4. Furthermore, an eight-step serial dilution SlipChip with a dilution ratio of 1:4 is applied for digital loop-mediated isothermal amplification (LAMP) across a large dynamic range and tested for hepatitis B viral load quantification with clinical samples. With 64 wells of each dilution and fewer than 600 wells in total, the serial dilution SlipChip can achieve a theoretical quantification dynamic range of 7 orders of magnitude.
In almost any branch of chemistry or life sciences, it is often necessary to study the interaction between different components in a system by varying their respective concentrations in a systematic manner. Currently, many procedures for generating a series of samples of different solute concentration levels are still done manually by dilution. To address this issue, we present herein a highly automated linear concentration gradient generator based on centrifugal microfluidics. The operation of this device is based on the use of multi-layered microfluidics in which individual fluidic samples to be mixed together are stored and metered in their respective layers before finally being transfered to a mixing chamber. To demonstrate the operation of this scheme, we have used the device to conduct antimicrobial susceptibility testing (AST). DI water, ampicillin solution and E.coli suspension were loaded to the chambers in different layers firstly. As the device went through several rounds of spinning at different speeds, a series of metered dosages of ampicillin along a linear concentration gradient were introduced to the mixing chamber and mixed with E.coli automatically. By monitoring the spectral absorbance of the suspensions, we were able to establish the minimum inhibitory concentration (MIC) value of ampicillin against E.coli. The process took about 3 hours to complete, and the experimental results showed a strong correlation with those obtained with standard CLSI broth dilution method. Clearly, the platform is useful for a wide range of applications such as drug discovery and personalised medicine, where concentration gradients are of concern.
Two-dimensional (2D) microdroplet arrays with indexed sample concentration gradients have been receiving considerable attention for high-throughput biological and medical analyses. However, the preparation of such an array by conventional methods mandates precise pipetting and/or pumping. In this paper, we introduce a method to spontaneously generate 2D-arrayed aqueous droplets using a well array, for which coarse pipetting is sufficient. The wells are connected in rows and columns via narrow channels. Aqueous solutions impregnated in the well array are split into droplets in every single well as a subsequently introduced immiscible solvent self-propagates and divides the solution at the channels. A concentration gradient of the samples can be formed across the connected solution in the well array; once droplets are generated, each droplet possesses a different sample concentration depending on its position in the array. We experimentally determined the optimal well dimensions and solvent species to obtain a high yield of droplet generation. We next demonstrated a 2D droplet array with a two-sample concentration gradient. Finally, the applicability of the system was demonstrated through a cell viability assay using a sample that induced apoptosis. We believe the proposed method contributes to simplification and miniaturization of the system to generate droplet arrays and thus is applicable to biological and medical analysis.
Rapid antimicrobial susceptibility testing (AST) is urgently needed for informing treatment decisions and preventing the spread of antimicrobial resistance resulting from the misuse and overuse of antibiotics. To date, no phenotypic AST exists that can be performed within a single patient visit (30 min) directly from clinical samples. We show that AST results can be obtained by using digital nucleic acid quantification to measure the phenotypic response of Escherichia coli present within clinical urine samples exposed to an antibiotic for 15 min. We performed this rapid AST using our ultrafast (~7 min) digital real-time loop-mediated isothermal amplification (dLAMP) assay [area under the curve (AUC), 0.96] and compared the results to a commercial (~2 hours) digital polymerase chain reaction assay (AUC, 0.98). The rapid dLAMP assay can be used with SlipChip microfluidic devices to determine the phenotypic antibiotic susceptibility of E. coli directly from clinical urine samples in less than 30 min. With further development for additional pathogens, antibiotics, and sample types, rapid digital AST (dAST) could enable rapid clinical decision-making, improve management of infectious diseases, and facilitate antimicrobial stewardship.
Significance
Antibiotic resistance is fueled by antibiotic misuse and has become a major global health concern. The phenomenon warrants improved diagnostics that can more rapidly and efficiently elucidate information about the infectious agent to aid in establishing a more targeted and knowledge-based treatment regimen. This paper introduces a rapid antibiotic susceptibility test and automated data analysis algorithm that can, unlike traditional methods, deliver results on the same working day in an efficient and translatable manner for clinical use. This paper also introduces a method for direct urine testing that can help save days of diagnosis time. The platform is expected to promote more judicious use of antibiotics, thereby reducing the emergence of antibiotic resistance, lowering healthcare costs and ultimately saving lives.
There remains an urgent need for rapid diagnostic methods that can evaluate antibiotic resistance for pathogenic bacteria in order to deliver targeted antibiotic treatments. Toward this end, we present a rapid and integrated single-cell biosensing platform, termed dropFAST, for bacterial growth detection and antimicrobial susceptibility assessment. DropFAST utilizes a rapid resazurin-based fluorescent growth assay coupled with stochastic confinement of bacteria in 20 pL droplets to detect signal from growing bacteria after 1h incubation, equivalent to 2-3 bacterial replications. Full integration of droplet generation, incubation, and detection into a single, uninterrupted stream also renders this platform uniquely suitable for in-line bacterial phenotypic growth assessment. To illustrate the concept of rapid digital antimicrobial susceptibility assessment, we employ the dropFAST platform to evaluate the antibacterial effect of gentamicin on E. coli growth.
Bacterial resistance to antimicrobial compounds is increasing at a faster rate than the development of new antibiotics; this represents a critical challenge for clinicians worldwide. Normally, the minimum inhibitory concentration of an antibiotic, the dosage at which bacterial growth is thwarted, provides an effective quantitative measure for antimicrobial susceptibility testing, and determination of minimum inhibitory concentration is conventionally performed by either a serial broth dilution method or with the commercially available Etest® (Biomerieux, France) kit. However, these techniques are relatively labor-intensive and require a significant amount of training. In order to reduce human error and increase operation simplicity, a simple microfluidic device that can perform antimicrobial susceptibility testing automatically via a broth dilution method to accurately determine the minimum inhibitory concentration was developed herein. As a proof of concept, wild-type (ATCC 29212) and vancomycin-resistant Enterococcus cells were incubated at five different vancomycin concentrations on-chip, and the sample injection, transport, and mixing processes occurred within five reaction chambers and three reagent chambers via the chip's automatic dispensation and dilution functions within nine minutes. The minimum inhibitory concentration values measured after 24 h of antibiotic incubation were similar to those calculated using Etest®. With its high flexibility, reliability, and portability, the developed microfluidic device provides a simple method for antimicrobial susceptibility testing in an automated format that could be implemented for clinical and point-of-care applications.
A smart multi-pipette for hand-held operation of microfluidic devices is presented and applied to cytotoxicity assays and micro-droplet generation. This method enables a continuous-flow and accurate pumping simply by pushing the plunger of the smart multi-pipette, thereby obviating the need for auxiliary equipment and special expertise in microfluidics. We applied the smart multi-pipette to a cytotoxicity assay using a gradient-generating device and water droplet generation using a T-junction device. In combination with general microfluidic devices, the smart multi-pipette enables the devices to successfully perform their own functions.
We describe a novel approach for generating a series of droplets with concentration gradient spanning 3-4 orders of magnitude by coupling flow injection gradient technique with droplet-based microfluidics. The present system was applied in enzyme inhibition assay to demonstrate its potential in high throughput drug screening. An enzyme inhibition curve spanning 3 orders of magnitude of the inhibitor concentration could be obtained with only a single 15-nL injection. Such a feature is particularly valuable for high-throughput screening with scarce samples or reagents.
Unlabelled:
From the first microfluidic devices used for analysis of single metabolic by-products to highly complex multicompartmental co-culture organ-on-chip platforms, efforts of many multidisciplinary teams around the world have been invested in overcoming the limitations of conventional research methods in the biomedical field. Close spatial and temporal control over fluids and physical parameters, integration of sensors for direct read-out as well as the possibility to increase throughput of screening through parallelization, multiplexing and automation are some of the advantages of microfluidic over conventional, 2D tissue culture in vitro systems. Moreover, small volumes and relatively small cell numbers used in experimental set-ups involving microfluidics, can potentially decrease research cost. On the other hand, these small volumes and numbers of cells also mean that many of the conventional molecular biology or biochemistry assays cannot be directly applied to experiments that are performed in microfluidic platforms. Development of different types of assays and evidence that such assays are indeed a suitable alternative to conventional ones is a step that needs to be taken in order to have microfluidics-based platforms fully adopted in biomedical research. In this review, rather than providing a comprehensive overview of the literature on microfluidics, we aim to discuss developments in the field of microfluidics that can aid advancement of biomedical research, with emphasis on the field of biomaterials. Three important topics will be discussed, being: screening, in particular high-throughput and combinatorial screening; mimicking of natural microenvironment ranging from 3D hydrogel-based cellular niches to organ-on-chip devices; and production of biomaterials with closely controlled properties. While important technical aspects of various platforms will be discussed, the focus is mainly on their applications, including the state-of-the-art, future perspectives and challenges.
Statement of significance:
Microfluidics, being a technology characterized by the engineered manipulation of fluids at the submillimeter scale, offers some interesting tools that can advance biomedical research and development. Screening platforms based on microfluidic technologies that allow high-throughput and combinatorial screening may lead to breakthrough discoveries not only in basic research but also relevant to clinical application. This is further strengthened by the fact that reliability of such screens may improve, since microfluidic systems allow close mimicking of physiological conditions. Finally, microfluidic systems are also very promising as micro factories of a new generation of natural or synthetic biomaterials and constructs, with finely controlled properties.
Droplet microfluidics is enabling reactions at nano- and picoliter scale, resulting in faster and cheaper biological and chemical analyses. However, varying concentrations of samples on a drop-to-drop basis is still a challenging task in droplet microfluidics, primarily limited due to lack of control over individual droplets. In this paper, we report an on-chip microfluidic droplet dilution strategy using three-valve peristaltic pumps.
A rapid antibiotic susceptibility test (AST) is desperately needed in clinical settings for fast and appropriate antibiotic administration. Traditional ASTs, which rely on cell culture, are not suitable for urgent cases of bacterial infection and antibiotic resistance owing to their relatively long test times. We describe a novel AST called single-cell morphological analysis (SCMA) that can determine antimicrobial susceptibility by automatically analyzing and categorizing morphological changes in single bacterial cells under various antimicrobial conditions. The SCMA was tested with four Clinical and Laboratory Standards Institute standard bacterial strains and 189 clinical samples, including extended-spectrum β-lactamase-positive Escherichia coli and Klebsiella pneumoniae, imipenem-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococci from hospitals. The results were compared with the gold standard broth microdilution test. The SCMA results were obtained in less than 4 hours, with 91.5% categorical agreement and 6.51% minor, 2.56% major, and 1.49% very major discrepancies. Thus, SCMA provides rapid and accurate antimicrobial susceptibility data that satisfy the recommended performance of the U.S. Food and Drug Administration.
Copyright © 2014, American Association for the Advancement of Science.
Significance
Obtaining cultures of microbes is essential for developing knowledge of bacterial genetics and physiology, but many microbes with potential biomedical significance identified from metagenomic studies have not yet been cultured due to the difficulty of identifying growth conditions, isolation, and characterization. We developed a microfluidics-based, genetically targeted approach to address these challenges. This approach corrects sampling bias from differential bacterial growth kinetics, enables the use of growth stimulants available only in small quantities, and allows targeted isolation and cultivation of a previously uncultured microbe from the human cecum that belongs to the high-priority group of the Human Microbiome Project’s “Most Wanted” list. This workflow could be leveraged to isolate novel microbes and focus cultivation efforts on biomedically important targets.
Isolating microbes carrying genes of interest from environmental samples is important for applications in biology and medicine. However, this involves the use of genetic assays that often require lysis of microbial cells, which is not compatible with the goal of obtaining live cells for isolation and culture. This paper describes the design, fabrication, biological validation, and underlying physics of a microfluidic SlipChip device that addresses this challenge. The device is composed of two conjoined plates containing 1000 microcompartments, each comprising two juxtaposed wells, one on each opposing plate. Single microbial cells are stochastically confined and subsequently cultured within the microcompartments. Then, we split each microcompartment into two replica droplets, both containing microbial culture, and then controllably separate the two plates while retaining each droplet within each well. We experimentally describe the droplet retention as a function of capillary pressure, viscous pressure, and viscosity of the aqueous phase. Within each pair of replicas, one can be used for genetic analysis, and the other preserves live cells for growth. This microfluidic approach provides a facile way to cultivate anaerobes from complex communities. We validate this method by targeting, isolating, and culturing Bacteroides vulgatus, a core gut anaerobe, from a clinical sample. To date, this methodology has enabled isolation of a novel microbial taxon, representing a new genus. This approach could also be extended to the study of other microorganisms and even mammalian systems, and may enable targeted retrieval of solutions in applications including digital PCR, sequencing, single cell analysis, and protein crystallization.
This paper describes a simple, versatile method of generating gradients in composition in solution or on surfaces using microfluidic systems. This method is based on controlled diffusive mixing of species in solutions that are flowing laminarly, at low Reynolds number, inside a network of microchannels. We demonstrate the use of this procedure to generate (1) gradients in the compositions of solutions, measured directly by colorimetric assays and (2) gradients in topography of the surfaces produced by generating concentration gradients of etching reagents, and then using these gradients to etch profiles into the substrate. The lateral dimensions of the gradients examined here, which went from 350 to 900 μm, are determined by the width of the microchannels. Gradients of different size, resolution, and shape have been generated using this method. The shape of the gradients can be changed continuously (dynamic gradients) by varying the relative flow velocities of the input streams of fluids. The method is experimentally simple and highly adaptable, and requires no special equipment except for an elastomeric relief structure that can be readily prepared by rapid prototyping. This technique provides a new platform with which to study phenomena that depend on gradients in concentration, especially dynamic phenomena in cell biology (chemotaxis and haptotaxis) and surface chemistry (nucleation and growth of crystals, etching, and Marangoni effects).
We described a microfluidic chip-based system capable of generating droplet array with a large scale concentration gradient by coupling flow injection gradient technique with droplet-based microfluidics. Multiple modules including sample injection, sample dispersion, gradient generation, droplet formation, mixing of sample and reagents, and online reaction within the droplets were integrated into the microchip. In the system, nanoliter-scale sample solution was automatically injected into the chip under valveless flow injection analysis mode. The sample zone was first dispersed in the microchannel to form a concentration gradient along the axial direction of the microchannel and then segmented into a linear array of droplets by immiscible oil phase. With the segmentation and protection of the oil phase, the concentration gradient profile of the sample was preserved in the droplet array with high fidelity. With a single injection of 16 nL of sample solution, an array of droplets with concentration gradient spanning 3-4 orders of magnitude could be generated. The present system was applied in the enzyme inhibition assay of β-galactosidase to preliminarily demonstrate its potential in high throughput drug screening. With a single injection of 16 nL of inhibitor solution, more than 240 in-droplet enzyme inhibition reactions with different inhibitor concentrations could be performed with an analysis time of 2.5 min. Compared with multiwell plate-based screening systems, the inhibitor consumption was reduced 1000-fold.
This article describes plug-based microfluidic technology that enables rapid detection and drug susceptibility screening of bacteria in samples, including complex biological matrices, without pre-incubation. Unlike conventional bacterial culture and detection methods, which rely on incubation of a sample to increase the concentration of bacteria to detectable levels, this method confines individual bacteria into droplets nanoliters in volume. When single cells are confined into plugs of small volume such that the loading is less than one bacterium per plug, the detection time is proportional to plug volume. Confinement increases cell density and allows released molecules to accumulate around the cell, eliminating the pre-incubation step and reducing the time required to detect the bacteria. We refer to this approach as 'stochastic confinement'. Using the microfluidic hybrid method, this technology was used to determine the antibiogram - or chart of antibiotic sensitivity - of methicillin-resistant Staphylococcus aureus (MRSA) to many antibiotics in a single experiment and to measure the minimal inhibitory concentration (MIC) of the drug cefoxitin (CFX) against this strain. In addition, this technology was used to distinguish between sensitive and resistant strains of S. aureus in samples of human blood plasma. High-throughput microfluidic techniques combined with single-cell measurements also enable multiple tests to be performed simultaneously on a single sample containing bacteria. This technology may provide a method of rapid and effective patient-specific treatment of bacterial infections and could be extended to a variety of applications that require multiple functional tests of bacterial samples on reduced timescales.
Pipetting and dilution are universal processes used in chemical and biological laboratories to assay and experiment. In microfluidics such operations are equally in demand, but difficult to implement. Recently, droplet-based microfluidics has emerged as an exciting new platform for high-throughput experimentation. However, it is challenging to vary the concentration of droplets rapidly and controllably. To this end, we developed a dilution module for high-throughput screening using droplet-based microfluidics. Briefly, a nanolitre-sized sample droplet of defined concentration is trapped within a microfluidic chamber. Through a process of droplet merging, mixing and re-splitting, this droplet is combined with a series of smaller buffer droplets to generate a sequence of output droplets that define a digital concentration gradient. Importantly, the formed droplets can be merged with other reagent droplets to enable rapid chemical and biological screens. As a proof of concept, we used the dilutor to perform a high-throughput homogeneous DNA-binding assay using only nanolitres of sample.
In this paper, we propose a microfluidic device that is capable of generating a concentration gradient followed by parallel droplet formation within channels with a simple T-junction geometry. Linear concentration gradient profiles can be obtained based on fluid diffusion under laminar flow. Optimized conditions for generating a linear concentration gradient and parallel droplet formation were investigated using fluorescent dye. The concentration gradient profile under diffusive mixing was dominated by the flow rate at sample inlets, while parallel droplet formation was affected by the channel geometry at both the inlet and outlet. The microfluidic device was experimentally characterized using optimal layout and operating conditions selected through a design process. Furthermore, in situ enzyme kinetic measurements of the β-galactosidase-catalyzed hydrolysis of resorufin-β-d-galactopyranoside were performed to demonstrate the application potential of our simple, time-effective, and low sample volume microfluidic device. We expect that, in addition to enzyme kinetics, drug screening and clinical diagnostic tests can be rapidly and accurately performed using this droplet-based microfluidic system.
To obtain protein crystals, researchers must search for conditions in multidimensional chemical space. Empirically, thousands of crystallization experiments are carried out to screen various precipitants at multiple concentrations. Microfluidics can manipulate fluids on a nanoliter scale, and it affects crystallization twofold. First, it miniaturizes the experiments that can currently be done on a larger scale and enables crystallization of proteins that are available only in small amounts. Second, it offers unique experimental approaches that are difficult or impossible to implement on a larger scale. Ongoing development of microfluidic techniques and their integration with protein production, characterization, and in situ diffraction promises to accelerate the progress of structural biology.
This paper describes two SlipChip-based approaches to protein crystallization: a SlipChip-based free interface diffusion (FID) method and a SlipChip-based composite method that simultaneously performs microbatch and FID crystallization methods in a single device. The FID SlipChip was designed to screen multiple reagents, each at multiple diffusion equilibration times, and was validated by screening conditions for crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis, against 48 different reagents at five different equilibration times each, consuming 12 microL of each protein for a total of 480 experiments using three SlipChips. The composite SlipChip was designed to screen multiple reagents, each at multiple mixing ratios and multiple equilibration times, and was validated by screening conditions for crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis. To prevent cross-contamination while keeping the solution in the neck channels for FID stable, the plates of the SlipChip were etched with a pattern of nanowells. This nanopattern was used to increase the contact angle of aqueous solutions on the surface of the silanized glass. The composite SlipChip increased the number of successful crystallization conditions and identified more conditions for crystallization than separate FID and microbatch screenings. Crystallization experiments were scaled up in well plates using conditions identified during the SlipChip screenings, and X-ray diffraction data were obtained to yield the protein structure of dihydrofolate reductase/thymidylate synthase at 1.95 A resolution. This free-interface diffusion approach provides a convenient and high-throughput method of setting up gradients in microfluidic devices and may find additional applications in cell-based assays.