A multiplexed nanoliter array-based microfluidic platform for quick, automatic antimicrobial susceptibility testing.

Antimicrobial resistance stemming from indiscriminate usage of antibiotics has emerged as a global healthcare issue with substantial economic implications. The inefficacy of commonly used antibiotics combined with superfluous consumption has worsened the issue. Rapid antimicrobial susceptibility testing (AST) to antibiotics can be advantageous in thwarting bacterial infections. Therefore, this study developed a simple nanoliter array-based microfluidic platform for performing rapid AST, which can handle and manipulate liquids both in nanoliter and microliter volumes. The platform consisted of two microfluidic devices, one for performing AST and another for diluting antibiotics and these two were suitably integrated. The microfluidic device used for generating microarrays for AST experiments is single-layered (no air layer) and has no active microvalves and air hole, which makes the device easy to fabricate and use. The loading process ensures uniform distribution of bacteria and relies on displacing the air from microarrays through porous polydimethylsiloxane membranes. Furthermore, the chip for dilution consisted of active microfluidic components, and could prepare and test seven different concentrations of antibiotics, which make the platform multiplexed and be capable of evaluating minimum inhibitory concentrations (MICs), a clinically relevant parameter. MIC determination requires less number of bacteria (∼2000) and hence shortens the pre-culture step, i.e. bacteria culture in blood and urine. This automated system demonstrated AST and evaluated MICs using Escherichia coli and two antibiotics, including ampicillin and streptomycin, and the results were ascertained using a gold standard method. It only took 8-9 h to perform AST, which is substantially less compared to a conventional process and hence is of high clinical utility.

An automated and portable antimicrobial susceptibility testing system for urinary tract infections.

Urinary tract infections (UTIs) are bacterial infections that 1) commonly affect females, 2) can pose high risks to impair kidney function, 3) are often treated with broad-spectrum antibiotics, and 4) are associated with high recurrence rates due to the evolution of drug-resistant strains. To choose the appropriate antibiotic, the minimum inhibitory concentration (MIC) among a panel of antibiotics should be determined before administration to avoid inadequate dosing or use of wrong antibiotics. To meet with the unmet needs, we developed a bead-based method for bacterial preconcentration with capture rates ranging from 20-50% and then automatically performed on-chip AST on an automated device which was composed of a pneumatic control module, a temperature control module and a chip image processing module. The developed portable system was capable of automatically conducting AST and MIC measurements using urine samples (via image analysis) in only 4.5-9 h and tested on four common UTIs bacterial strains. This compact system may therefore be promising for point-of-care personalized medicine in the near future.

Rapid antimicrobial susceptibility tests on an integrated microfluidic device for precision medicine of antibiotics.

This study reports an integrated microfluidic device that was capable of executing rapid antimicrobial susceptibility tests with one, two, or even three antibiotics against two clinically isolated multi-drug-resistant bacteria strains (including carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus). Bacteria were automatically mixed for 10 min with serially diluted antibiotics with a novel, membrane-type micromixer consisting of two circular micropumps, and the minimum inhibitory concentrations (MIC) were then determined via simple colorimetric reactions in only 4.5-6 h using only 3 µL of bacteria sample of each reaction (as opposed to 24 h and 50 µL, respectively, with the conventional broth micro-dilution method). In addition to determining MICs of antibiotics (ceftazidime, gentamicin, meropenem, vancomycin and linezolid), interaction effects across antibiotics combinations (gentamicin/meropenem or ceftazidime/gentamicin/meropenem) at different dosages were explored. The efficacy of polypharmacy showed additivity when gentamicin or ceftazidime/gentamicin were combined with meropenem to treat carbapenem-resistant Escherichia coli. This represents the first time that the perplexing clinical decision to choose multiple antibiotics for combination therapy against drug resistant bacteria can be realized on an integrated microfluidic device within 6 h.

An integrated microfluidic system for rapid, automatic and high-throughput staining of clinical tissue samples for diagnosis of ovarian cancer.

Accurate cancer diagnostic methods are of urgent need. Since traditional immunohistochemistry (IHC)-based approaches, while reliable, are labor-intensive and require well-trained technicians, we developed an integrated microfluidic platform capable of labeling ovarian cancer biomarkers (i.e. aptamer) within formalin-fixed, paraffin embedded tissues via molecular probes. Both aptamer-based 1) fluorescent staining and 2) IHC staining of clinical tissue samples could be automated in the microfluidic system in only 2-3 h (40-50% faster than conventional approaches) with <0.5 mL of reagents, signifying that this device could serve as a promising diagnostic tool for ovarian cancer.

An integrated microfluidic system for antimicrobial susceptibility testing with antibiotic combination.

Polypharmacy is routinely administered to fight severe infections, though it has led to rampant multi-drug resistance in many bacterial strains. Preferably, antimicrobial susceptibility testing (AST) would be carried out prior to antibiotic prescription, though it is generally thought to be too complex and labor-intensive. In order to assist clinicians with better antibiotic administration for the effective treatment of bacterial infections, an integrated microfluidic system (IMS) capable of automating AST for 1-2 antibiotics against clinical bacterial pathogens was developed herein. Accurate determination of the minimum and fractional inhibitory concentrations of vancomycin, gentamicin, and linezolid were determined by assaying growth of two clinical methicillin-resistant Staphylococcus aureus isolates via a colorimetric assay on-chip. By applying various antibiotic combinations against a single pathogen in multiple chambers, the IMS could identify the optimal drug combination and the minimum effective dosage by evaluating the fractional inhibitory concentration index. This IMS possessed several advantages over conventional methods, including (1) a 50% reduction in bacterial sample and reagent volume (<50 µL per well), (2) less potential for human error due to its automatic nature, (3) faster liquid manipulation time by integrating the microfluidic components rather than labor-intensive process, and (4) straightforward result interpretation via colorimetric change instead of turbidity degree. Personalized medicine for treatment of bacterial infections may therefore be realized using this IMS.

Generating digital drug cocktails via optical manipulation of drug-containing particles and photo-patterning of hydrogels.

An integrated microfluidic system combining 1) an optically-induced-dielectrophoresis (ODEP) module for manipulation of drug-containing particles and 2) an ultraviolet (UV) "direct writing" module capable of patterning hydrogels was established herein for automatic formulation of customized digital drug cocktails. Using the ODEP module, the drug-containing particles were assembled by using moving light patterns generated from a digital projector. The hydrogel, poly(ethylene glycol) diacrylate (PEGDA), was used as the medium in the ODEP module such that the assembled drug-containing particles could be UV-cured and consequently encapsulated in "pills" of specific sizes and shapes by using the UV direct writing module. At an optimal ODEP force of 335 pN, which was achieved in a solution of 15% PEGDA in 0.2 M sucrose, it was possible to manipulate and UV-cure the drug-containing particles. Furthermore, with a digital micromirror device inside the UV direct writing module, different UV patterns could be designed and projected, allowing for the digital drug cocktails to be packaged into different shapes in <60 s. As a demonstration, emulsion droplets containing two different anti-cancer drugs were further tested to show the capability of the developed device. This represents an automatic digital drug cocktail formulating device which stands to revolutionize personalized medicine.

An automated microfluidic system for selection of aptamer probes against ovarian cancer tissues.

Because of the difficulty of treatment in its latest stages, cancer is among the leading causes of death worldwide. Therefore, high-affinity and specificity biomarkers are still in demand for many cancer types, and the utility of aptamers to serve in this regard has been explored recently. Although a process known as "systematic evolution of ligands by exponential enrichment" (SELEX) has been used to generate aptamer-based cancer biomarkers, this approach is complicated, time-consuming, and labor-intensive. An automated microfluidic system was consequently developed herein to screen ovarian cancer-specific aptamers via on-chip SELEX with clinical cancer tissue samples. The integrated microfluidic system consisted of an integrated microfluidic chip, a temperature control module equipped with 12 thermoelectric coolers, and a flow control module for controlling 36 electromagnetic valves such that the entire, tissue-based SELEX process could be fully automated and carried out within 15 h. Highly specific ovarian cancer aptamers with high affinity (dissociation constant of 129 nM) to their cellular targets were screened with this system. Given the comparable specificity to their much more expensive antibody counterparts, these aptamers, when used in conjunction with the developed microfluidic system, may be used to diagnose ovarian cancer in its earliest stages.

Automatic cell fusion via optically-induced dielectrophoresis and optically-induced locally-enhanced electric field on a microfluidic chip.

Cell fusion technology has been exploited in a wide variety of biomedical applications, and physical, chemical, and biological approaches can all be used to fuse two different types of cells; however, no current technique is adept at inducing both cell pairing and fusion at high efficiencies and yields. Hence, we developed a new method featuring the use of optically induced dielectrophoresis (ODEP) in conjunction with an optically induced, locally enhanced electric field for accurate and automatic cell pairing and fusion on a microfluidic device. After pairing cells via ODEP, a locally enhanced electric field generated by "virtual electrodes" by projecting light patterns was enacted to induce a proper transmembrane potential at the cell contact area such that cell fusion could be triggered by white light exposure. As a fusion yield of 9.67% was achieved between Pan1 and A549 cells, we believe that this may be a promising technique for automatically fusing different cell types.

A microfluidic device for antimicrobial susceptibility testing based on a broth dilution method.

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 24h 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.

Light-RCV: a lightweight read coverage viewer for next generation sequencing data.

BACKGROUND: Next-generation sequencing (NGS) technologies has brought an unprecedented amount of genomic data for analysis. Unlike array-based profiling technologies, NGS can reveal the expression profile across a transcript at the base level. Such a base-level read coverage provides further insights for alternative mRNA splicing, single-nucleotide polymorphism (SNP), novel transcript discovery, etc. However, to our best knowledge, none of existing NGS viewers can timely visualize genome-wide base-level read coverages in an interactive environment. RESULTS: This study proposes an efficient visualization pipeline and implements a lightweight read coverage viewer, Light-RCV, with the proposed pipeline. Light-RCV consists of four featured designs on the path from raw NGS data to the final visualized read coverage: i) read coverage construction algorithm, ii) multi-resolution profiles, iii) two-stage architecture and iv) storage format. With these designs, Light-RCV achieves a < 0.5s response time on any scale of genomic ranges, including whole chromosomes. Finally, a case study was performed to demonstrate the importance of visualizing base-level read coverage and the value of Light-RCV. CONCLUSIONS: Compared with multi-functional genome viewers such as Artemis, Savant, Tablet and Integrative Genomics Viewer (IGV), Light-RCV is designed only for visualization. Therefore, it does not provide advanced analyses. However, its backend technology provides an efficient kernel of base-level visualization that can be easily embedded to other viewers. This viewer is the first to provide timely visualization of genome-wide read coverage at the base level in an interactive environment. The software is available for free at http://lightrcv.ee.ncku.edu.tw.

Optimizing information in Next-Generation-Sequencing (NGS) reads for improving de novo genome assembly.

Next-Generation-Sequencing is advantageous because of its much higher data throughput and much lower cost compared with the traditional Sanger method. However, NGS reads are shorter than Sanger reads, making de novo genome assembly very challenging. Because genome assembly is essential for all downstream biological studies, great efforts have been made to enhance the completeness of genome assembly, which requires the presence of long reads or long distance information. To improve de novo genome assembly, we develop a computational program, ARF-PE, to increase the length of Illumina reads. ARF-PE takes as input Illumina paired-end (PE) reads and recovers the original DNA fragments from which two ends the paired reads are obtained. On the PE data of four bacteria, ARF-PE recovered >87% of the DNA fragments and achieved >98% of perfect DNA fragment recovery. Using Velvet, SOAPdenovo, Newbler, and CABOG, we evaluated the benefits of recovered DNA fragments to genome assembly. For all four bacteria, the recovered DNA fragments increased the assembly contiguity. For example, the N50 lengths of the P. brasiliensis contigs assembled by SOAPdenovo and Newbler increased from 80,524 bp to 166,573 bp and from 80,655 bp to 193,388 bp, respectively. ARF-PE also increased assembly accuracy in many cases. On the PE data of two fungi and a human chromosome, ARF-PE doubled and tripled the N50 length. However, the assembly accuracies dropped, but still remained >91%. In general, ARF-PE can increase both assembly contiguity and accuracy for bacterial genomes. For complex eukaryotic genomes, ARF-PE is promising because it raises assembly contiguity. But future error correction is needed for ARF-PE to also increase the assembly accuracy. ARF-PE is freely available at http://140.116.235.124/~tliu/arf-pe/.

Robustness and topology of the yeast cell cycle Boolean network.

Yeast cell cycle Boolean network was used as a case study of robustness to protein noise. Robustness was interpreted as involving stability of G1 steady state and sequence of gene expression from cell cycle START to stationary G1. A robustness measure to evaluate robustness strength of a network was proposed. Robust putative networks corresponding to the same steady state and sequence of gene expression of wild-type network were sampled. Architecture of wild-type yeast cell cycle network can be revealed by average topology profile of sampled robust putative networks.

Biomedical microdevices synthesis of iron oxide nanoparticles using a microfluidic system.

The preparation of nanoparticles is essential in the application of many nanotechnologies and various preparation methods have been explored in the previous decades. Among them, iron oxide nanoparticles have been widely investigated in applications ranging from bio-imaging to bio-sensing due to their unique magnetic properties. Recently, microfluidic systems have been utilized for synthesis of nanoparticles, which have the advantages of automation, well-controlled reactions, and a high particle uniformity. In this study, a new microfluidic system capable of mixing, transporting and reacting was developed for the synthesis of iron oxide nanoparticles. It allowed for a rapid and efficient approach to accelerate and automate the synthesis of the iron oxide nanoparticles as compared with traditional methods. The microfluidic system uses micro-electro-mechanical-system technologies to integrate a new double-loop micromixer, two micropumps, and a microvalve on a single chip. When compared with large-scale synthesis systems with commonly-observed particle aggregation issues, successful synthesis of dispersed and uniform iron oxide nanoparticles has been observed within a shorter period of time (15 min). It was found that the size distribution of these iron oxide nanoparticles is superior to that of the large-scale systems without requiring any extra additives or heating. The size distribution had a variation of 16%. This is much lower than a comparable large-scale system (34%). The development of this microfluidic system is promising for the synthesis of nanoparticles for many future biomedical applications.