Digital microfluidics as an emerging tool for bacterial protocols.

Bacteria are widely studied in various research areas, including synthetic biology, sequencing and diagnostic testing. Protocols involving bacteria are often multistep, cumbersome and require access to a long list of instruments to perform experiments. In order to streamline these processes, the fluid handling technique digital microfluidics (DMF) has provided a miniaturized platform to perform various steps of bacterial protocols from sample preparation to analysis. DMF devices can be paired/interfaced with instrumentation such as microscopes, plate readers, and incubators, demonstrating their versatility with existing research tools. Alternatively, DMF instruments can be integrated into all-in-one packages with on-chip magnetic separation for sample preparation, heating/cooling modules to perform assay steps and cameras for absorbance and/or fluorescence measurements. This perspective outlines the beneficial features DMF offers to bacterial protocols, highlights limitations of current work and proposes future directions for this tool's expansion in the field.

Bacterial classification and antibiotic susceptibility testing on an integrated microfluidic platform.

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.

Multifunctional 3D-Printed Wound Dressings.

Personalized wound dressings provide enhanced healing for different wound types; however multicomponent wound dressings with discretely controllable delivery of different biologically active agents are yet to be developed. Here we report 3D-printed multicomponent biocomposite hydrogel wound dressings that have been selectively loaded with small molecules, metal nanoparticles, and proteins for independently controlled release at the wound site. Hydrogel wound dressings carrying antibacterial silver nanoparticles and vascular endothelial growth factor with predetermined release profiles were utilized to study the physiological response of the wound in a mouse model. Compared to controls, the application of dressings resulted in improvement in granulation tissue formation and differential levels of vascular density, dependent on the release profile of the growth factor. Our study demonstrates the versatility of the 3D-printed hydrogel dressings that can yield varied physiological responses in vivo and can further be adapted for personalized treatment of various wound types.

Fluorescent Droplet Cytometry for On-Cell Phenotype Tracking.

Profiling the heterogeneous phenotypes of live cancer cells is a key capability that requires single-cell analysis. However, acquiring information at the single-cell level for live cancer cells is challenging when small collections of cells are being targeted. Here, we report single-cell analysis for low abundance cells enabled by fluorescent droplet cytometry (FDC), an approach that uses a biomarker-specific enzymatic fluorescent assay carried out using a droplet microfluidic platform. FDC utilizes DNA-functionalized antibodies in droplets to achieve specific on-cell target detection and enables characterization and profiling of live cancer cells with single-cell resolution based on their surface phenotype. Using this approach, we achieve live-cell phenotypic profiling of multiple surface markers acquired with small (<40 cells) collections of cells.

Magnetic Ranking Cytometry: Profiling Rare Cells at the Single-Cell Level.

Cellular heterogeneity in biological systems presents major challenges in the diagnosis and treatment of disease and also complicates the deconvolution of complex cellular phenomena. Single-cell analysis methods provide information that is not masked by the intrinsic heterogeneity of the bulk population and can therefore be applied to gain insights into heterogeneity among different cell subpopulations with fine resolution. Over the last 5 years, an explosion in the number of single-cell measurement methods has occurred. However, most of these methods are applicable to pure populations of cultured cells and are not able to handle high levels of phenotypic heterogeneity or a large background of nontarget cells. Microfluidics is an attractive tool for single cell manipulation as it enables individual encasing of single cells, allowing for high-throughput analysis with precise control of the local environment. Our laboratory has developed a new microfluidics-based analytical strategy to meet this unmet need referred to as magnetic ranking cytometry (MagRC). Cells expressing a biomarker of interest are labeled with receptor-coated magnetic nanoparticles and isolated from nontarget cells using a microfluidic device. The device ranks the cells according to the level of bound magnetic nanoparticles, which corresponds to the expression level of a target biomarker. Over the last several years, two generations of MagRC devices have been developed for different applications. The first-generation MagRC devices are powerful tools for the quantitation and analysis of rare cells present in heterogeneous samples, such as circulating tumor cells, stem cells, and pathogenic bacteria. The second-generation MagRC devices are compatible with the efficient recovery of cells sorted on the basis of protein expression and can be used to analyze large populations of cells and perform phenotypic CRISPR screens. To improve analytical precision, newer iterations of the first-generation and second-generation MagRC devices have been integrated with electrochemical sensors and Hall effect sensors, respectively. Both generations of MagRC devices permit the isolation of viable cells, which sets the stage for a wide range of applications, such as generating cell lines from rare cells and in vitro screening for effective therapeutic interventions in cancer patients to realize the promise of personalized medicine. This Account summarizes the development and application of the MagRC and describes a suite of advances that have enabled single-cell tumor cell analysis and monitoring tumor response to therapy, stem cell analysis, and detection of pathogens.

Characterization of the Human Skeletal Muscle Metabolome for Elucidating the Mechanisms of Bicarbonate Ingestion on Strenuous Interval Exercise.

Bicarbonate has long been touted as a putative ergogenic aid that improves exercise performance and blood buffering capacity during strenuous exercise. However, the underlying mechanisms of action of bicarbonate intake on skeletal muscle metabolism have yet to be fully elucidated. Herein, we apply two orthogonal analytical platforms for nontargeted profiling of metabolites and targeted analysis of electrolytes from mass-limited muscle tissue biopsies (∼2 mg dried mass) when multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) and CE with indirect UV detection are used, respectively. Seven untrained men performed a standardized bout of high-intensity interval exercise trial following either bicarbonate (0.40 g/kg) or placebo ingestion in a double-blinded, placebo-controlled, crossover study design, where paired skeletal muscle tissue and plasma specimens were collected at three time intervals at rest, postexercise, and recovery. Optimization of a quantitative microextraction procedure was first developed for lyophilized tissue prior to characterization of the human muscle metabolome, which resulted in the identification and quantification of more than 80 polar/ionic metabolites reliably (CV < 30%) detected in a majority (>75%) of samples with quality control. Complementary univariate and multivariate statistical methods were used to identify biomarkers associated with strenuous exercise and/or bicarbonate treatment responses, whereas structural elucidation of biologically significant intramuscular metabolites was performed using high-resolution MS/MS. Importantly, bicarbonate ingestion prior to strenuous interval exercise was found to elicit a modest treatment effect ( p < 0.05) in comparison to placebo on metabolic pathways associated with ionic homeostasis (potassium), purine degradation (uric acid), and oxidative stress as regulated by glutathione metabolism (oxidized mixed glutathione disulfide) and histidine-containing dipeptides (anserine) within muscle tissue that was distinctive from dynamic metabolic changes measured in circulation. This work provides deeper biochemical insights into the effect of acute alkalosis in preserving contracting muscle function during high-intensity exercise, which is also applicable to the study of muscle-related pathologies relevant to human health and aging.

Nanoparticle-Mediated Capture and Electrochemical Detection of Methicillin-Resistant Staphylococcus aureus.

The spread of antibiotic-resistant bacteria poses a global threat to public health. Conventional bacterial detection and identification methods often require pre-enrichment and/or sample preprocessing and purification steps that can prolong diagnosis by days. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most widespread antibiotic-resistant bacteria and is the leading cause of hospital-acquired infections. Here, we have developed a method to specifically capture and detect MRSA directly from patient nasal swabs with no prior culture and minimal processing steps using a microfluidic device and antibody-functionalized magnetic nanoparticles. Bacteria are captured based on antibody recognition of a membrane-bound protein marker that confers ß-lactam antibiotic resistance. MRSA identification is then achieved by the use of a strain-specific antibody functionalized with alkaline phosphatase for electrochemical detection. This approach ensures that only those bacteria of the target strain and resistance profile are measured. The method has a limit of detection of 845 CFU/mL and excellent discrimination against high concentrations of common nontarget nasal flora with a turnaround time of under 4.5 h. This detection method was successfully validated using clinical nasal swab specimens ( n = 30) and has the potential to be tailored to various bacterial targets.

Curvature-Mediated Surface Accessibility Enables Ultrasensitive Electrochemical Human Methyltransferase Analysis.

The development of new tools for tracking the activity of human DNA methyltransferases is an important goal given the role of this enzyme as a cancer biomarker and epigenetic modulator. However, analysis of the human DNA (cytosine-5)-methyltransferase 1 (Dnmt1) activity is challenging, especially in crude samples, because of the low activity and large size of the enzyme. Here, we report a new approach to Dnmt analysis that combines nanostructured electrodes with a digest-and-amplify strategy that directly monitors Dnmt1 activity with high sensitivity. Nanostructured electrodes are required for the function of the assay to promote the accessibility of the electrode for human Dnmt1. Moreover, DNA-templated deposition of silver nanoparticles (for signal amplification) is combined with DNA Exonuclease I digestion to yield optimal target-to-control signals. We achieve high sensitivity for the detection of human Dnmt1, and particularly Dnmt1 from crude cell lysates. Specifically, the detection limit of our electrochemical assay is 20 pM, which is 2 orders of magnitude lower than previously reported methods. In crude lysates, we detected Dnmt1 from as few as five colorectal cancer cells (HCT116). With biopsy samples, we were able to distinguish colorectal tumor tissue from healthy adjacent tissue using only 10 µg of sample. The strategy enables analysis of an important marker underlying the epigenetic basis of cancerous transformation.

Tunability of liquid-infused silicone materials for biointerfaces.

The ability to control the properties of bio-inspired liquid-infused surfaces is of interest in a wide range of applications. Liquid layers created using oil-infused polydimethylsiloxane elastomers offer a potentially simple way of accomplishing this goal through the adjustment of parameters such as curing agent ratio and oil viscosity. In this work, the effect of tuning these compositional parameters on the properties of the infused polymer are investigated, including infusion dynamics, stiffness, longevity in the face of continuous liquid overlayer removal, and resistance to bacterial adhesion. It is found that that curing agent concentration appears to have the greatest impact on the functionality of the system, with a lower base-to-curing agent ratio resulting in both increased longevity and improved resistance to adhesion by Escherichia coli. A demonstration of how these findings may be implemented to introduce patterned wettability to the surface of the infused polymers is presented by controlling the spatial arrangement of bacteria. These results demonstrate a new degree of control over immobilized liquid layers and will facilitate their use in future applications.

A fully-integrated and automated testing device for PCR-free viral nucleic acid detection in whole blood.

Integrated devices for automated nucleic acid testing (NAT) are critical for infectious disease diagnosis to be performed outside of centralized laboratories. The gold standard methods for NAT are enzymatic amplification methods like the polymerase chain reaction that typically require expensive equipment and highly-trained personnel, limiting use in low-resource settings. A low-cost, integrated, rapid, portable and user-friendly point-of-care (POC) nucleic acid diagnostic device will improve the accessibility of NAT. Here, we present a fully integrated and simple-to-use POC device operated by a passive fluidic method that is able to perform a sequential multi-step assay to detect viral nucleic acids in blood. This simple device enabled the rapid detection of hepatitis C virus in blood in approximately 30 minutes with minimal sample handling by the user.

Chemistry-Driven Approaches for Ultrasensitive Nucleic Acid Detection.

Methods that can rapidly and specifically analyze nucleic acid sequences will revolutionize the diagnosis and treatment of disease by allowing molecular-level information to be used during routine medicine. In this Perspective, we discuss chemistry-driven approaches that will make the detection of DNA and RNA sequences more routine in clinical settings. In addition, we discuss unmet needs and areas where future effort is necessary to enable nucleic acids analysis to become a mainstream tool in routine clinical medicine. Methods for next-generation sequencing of DNA are producing a wealth of information by allowing the study of how specific genetic mutations or single nucleotide polymorphisms influence the onset of disease, prognosis, or response to treatment. To give this information clinical utility, new methods of detecting nucleic acid sequences are being developed in order to rapidly obtain genetic information in more streamlined formats, and with the ability to obtain information outside of a laboratory setting. Challenges remain in this area, however, and new chemistries that will facilitate fast, simple nucleic acids analysis in a clinical setting are needed.

Mitochondrial DNA damage by bleomycin induces AML cell death.

Mitochondria contain multiple copies of their own 16.6 kb circular genome. To explore the impact of mitochondrial DNA (mtDNA) damage on mitochondrial (mt) function and viability of AML cells, we screened a panel of DNA damaging chemotherapeutic agents to identify drugs that could damage mtDNA. We identified bleomycin as an agent that damaged mtDNA in AML cells at concentrations that induced cell death. Bleomycin also induced mtDNA damage in primary AML samples. Consistent with the observed mtDNA damage, bleomycin reduced mt mass and basal oxygen consumption in AML cells. We also demonstrated that the observed mtDNA damage was functionally important for bleomycin-induced cell death. Finally, bleomycin delayed tumor growth in xenograft mouse models of AML and anti-leukemic concentrations of the drug induced mtDNA damage in AML cells preferentially over normal lung tissue. Taken together, mtDNA-targeted therapy may be an effective strategy to target AML cells and bleomycin could be useful in the treatment of this disease.