Directed evolution of material-producing microorganisms.

Nature is home to a variety of microorganisms that create materials under environmentally friendly conditions. While this offers an attractive approach for sustainable manufacturing, the production of materials by native microorganisms is usually slow and synthetic biology tools to engineer faster microorganisms are only available when prior knowledge of genotype-phenotype links is available. Here, we utilize a high-throughput directed evolution platform to enhance the fitness of whole microorganisms under selection pressure and identify genetic pathways to enhance the material production capabilities of native species. Using Komagataeibacter sucrofermentans as a model cellulose-producing microorganism, we show that our droplet-based microfluidic platform enables the directed evolution of these bacteria toward a small number of cellulose overproducers from an initial pool of 40,000 random mutants. Sequencing of the evolved strains reveals an unexpected link between the cellulose-forming ability of the bacteria and a gene encoding a protease complex responsible for protein turnover in the cell. The ability to enhance the fitness of microorganisms toward a specific phenotype and to unravel genotype-phenotype links makes this high-throughput directed evolution platform a promising tool for the development of new strains for the sustainable manufacturing of materials.

In Situ Complexation of sgRNA and Cas12a Improves the Performance of a One-Pot RPA-CRISPR-Cas12 Assay.

Due to their ability to selectively target pathogen-specific nucleic acids, CRISPR-Cas systems are increasingly being employed as diagnostic tools. "One-pot" assays that combine nucleic acid amplification and CRISPR-Cas systems (NAAT-CRISPR-Cas) in a single step have emerged as one of the most popular CRISPR-Cas biosensing formats. However, operational simplicity comes at a cost, with one-pot assays typically being less sensitive than corresponding two-step NAAT-CRISPR-Cas assays and often failing to detect targets at low concentrations. It is thought that these performance reductions result from the competition between the two enzymatic processes driving the assay, namely, Cas-mediated cis-cleavage and polymerase-mediated amplification of the target DNA. Herein, we describe a novel one-pot RPA-Cas12a assay that circumvents this issue by leveraging in situ complexation of the target-specific sgRNA and Cas12a to purposefully limit the concentration of active Cas12a during the early stages of the assay. Using a clinically relevant assay against a DNA target for HPV-16, we show how this in situ format reduces competition between target cleavage and amplification and engenders significant improvements in detection limit when compared to the traditional one-pot assay format, even in patient-derived samples. Finally, to gain further insight into the assay, we use experimental data to formulate a mechanistic model describing the competition between the Cas enzyme and nucleic acid amplification. These findings suggest that purposefully limiting cis-cleavage rates of Cas proteins is a viable strategy for improving the performance of one-pot NAAT-CRISPR-Cas assays.

Association between atrial fibrillation and heart failure patient reported outcomes across the ejection fraction spectrum.

BACKGROUND: Atrial fibrillation (AF) is common in patients with heart failure (HF) and is associated with worse clinical outcomes. We evaluated the relationship between AF and longitudinal changes in health-related quality of life (HRQoL) measured by Kansas City Cardiomyopathy Questionnaire (KCCQ) in both HF with preserved (HFpEF) and reduced ejection fraction (HFrEF). METHODS: This is a post-hoc analysis of the TOPCAT and HF-ACTION trials. The effect of AF on KCCQ overall summary scores (OSS), in both trials, was examined using a mixed effects regression model. Patients were divided into 3 groups according to AF status at baseline: patients with a history of AF but no AF detected on ECG at enrollment (Hx AF group), patients with history of AF and AF detected on ECG at enrollment (ECG AF group) and patients with post-randomization new-onset AF (New AF group). RESULTS: In TOPCAT, among 1,710 patients with KCCQ data available, AF was associated with a significantly lower KCCQ-OSS (-3.98; 95% CI -7.21: -0.74) at 48 months, with a significant AF status by time interaction (P = .03). In HF-ACTION, among 1,814 patients with available KCCQ data, AF was associated with a significantly lower KCCQ-OSS (-3.67; 95% CI -6.21: -1.41) at 24 months but there was no significant AF status by time interaction. In both trials, the type of AF was not associated with significant changes in KCCQ-OSS score. CONCLUSION: Ιn patients with both HFpEF and HFrEF, AF was independently associated with worse HRQoL measured by KCCQ.

Synergistic Effects of Weight Loss and Catheter Ablation: Can microRNAs Serve as Predictive Biomarkers for the Prevention of Atrial Fibrillation Recurrence?

In atrial fibrillation (AF), multifactorial pathologic atrial alterations are manifested by structural and electrophysiological changes known as atrial remodeling. AF frequently develops in the context of underlying cardiac abnormalities. A critical mechanistic role played by atrial stretch is played by abnormal substrates in a number of conditions that predispose to AF, including obesity, heart failure, hypertension, and sleep apnea. The significant role of overweight and obesity in the development of AF is known; however, the differential effect of overweight, obesity, cardiovascular comorbidities, lifestyle, and other modifiable risk factors on the occurrence and recurrence of AF remains to be determined. Reverse remodeling of the atrial substrate and subsequent reduction in the AF burden by conversion into a typical sinus rhythm has been associated with weight loss through lifestyle changes or surgery. This makes it an essential pillar in the management of AF in obese patients. According to recently published research, microRNAs (miRs) may function as post-transcriptional regulators of genes involved in atrial remodeling, potentially contributing to the pathophysiology of AF. The focus of this review is on their modulation by both weight loss and catheter ablation interventions to counteract atrial remodeling in AF. Our analysis outlines the experimental and clinical evidence supporting the synergistic effects of weight loss and catheter ablation (CA) in reversing atrial electrical and structural remodeling in AF onset and in recurrent post-ablation AF by attenuating pro-thrombotic, pro-inflammatory, pro-fibrotic, arrhythmogenic, and male-sex-associated hypertrophic remodeling pathways. Furthermore, we discuss the promising role of miRs with prognostic potential as predictive biomarkers in guiding approaches to AF recurrence prevention.

Effect of Low-Level Tragus Stimulation on Cardiac Metabolism in Heart Failure with Preserved Ejection Fraction: A Transcriptomics-Based Analysis.

Abnormal cardiac metabolism precedes and contributes to structural changes in heart failure. Low-level tragus stimulation (LLTS) can attenuate structural remodeling in heart failure with preserved ejection fraction (HFpEF). The role of LLTS on cardiac metabolism is not known. Dahl salt-sensitive rats of 7 weeks of age were randomized into three groups: low salt (0.3% NaCl) diet (control group; n = 6), high salt diet (8% NaCl) with either LLTS (active group; n = 8), or sham stimulation (sham group; n = 5). Both active and sham groups received the high salt diet for 10 weeks with active LLTS or sham stimulation (20 Hz, 2 mA, 0.2 ms) for 30 min daily for the last 4 weeks. At the endpoint, left ventricular tissue was used for RNA sequencing and transcriptomic analysis. The Ingenuity Pathway Analysis tool (IPA) was used to identify canonical metabolic pathways and upstream regulators. Principal component analysis demonstrated overlapping expression of important metabolic genes between the LLTS, and control groups compared to the sham group. Canonical metabolic pathway analysis showed downregulation of the oxidative phosphorylation (Z-score: -4.707, control vs. sham) in HFpEF and LLTS improved the oxidative phosphorylation (Z-score = -2.309, active vs. sham). HFpEF was associated with the abnormalities of metabolic upstream regulators, including PPARGC1α, insulin receptor signaling, PPARα, PPARδ, PPARGC1ß, the fatty acid transporter SLC27A2, and lysine-specific demethylase 5A (KDM5A). LLTS attenuated abnormal insulin receptor and KDM5A signaling. HFpEF is associated with abnormal cardiac metabolism. LLTS, by modulating the functioning of crucial upstream regulators, improves cardiac metabolism and mitochondrial oxidative phosphorylation.

High-Throughput Absorbance-Activated Droplet Sorting for Engineering Aldehyde Dehydrogenases.

Recent decades have seen a dramatic increase in the commercial use of biocatalysts, transitioning from energy-intensive traditional chemistries to more sustainable methods. Current enzyme engineering techniques, such as directed evolution, require the generation and testing of large mutant libraries to identify optimized variants. Unfortunately, conventional screening methods are unable to screen such large libraries in a robust and timely manner. Droplet-based microfluidic systems have emerged as a powerful high-throughput tool for library screening at kilohertz rates. Unfortunately, almost all reported systems are based on fluorescence detection, restricting their use to a limited number of enzyme types that naturally convert fluorogenic substrates or require the use of surrogate substrates. To expand the range of enzymes amenable to evolution using droplet-based microfluidic systems, we present an absorbance-activated droplet sorter that allows of droplet sorting at kilohertz rates without the need for optical monitoring of the microfluidic system. To demonstrate the utility of the sorter, we rapidly screen a 105-member aldehyde dehydrogenase library towards D-glyceraldehyde using a NADH mediated coupled assay that generates WST-1 formazan as the colorimetric product. We successfully identify a variant with a 51% improvement in catalytic efficiency and a significant increase in overall activity across a broad substrate spectrum.

Consumer-Led Screening for Atrial Fibrillation: Frontier Review of the AF-SCREEN International Collaboration.

The technological evolution and widespread availability of wearables and handheld ECG devices capable of screening for atrial fibrillation (AF), and their promotion directly to consumers, has focused attention of health care professionals and patient organizations on consumer-led AF screening. In this Frontiers review, members of the AF-SCREEN International Collaboration provide a critical appraisal of this rapidly evolving field to increase awareness of the complexities and uncertainties surrounding consumer-led AF screening. Although there are numerous commercially available devices directly marketed to consumers for AF monitoring and identification of unrecognized AF, health care professional-led randomized controlled studies using multiple ECG recordings or continuous ECG monitoring to detect AF have failed to demonstrate a significant reduction in stroke. Although it remains uncertain if consumer-led AF screening reduces stroke, it could increase early diagnosis of AF and facilitate an integrated approach, including appropriate anticoagulation, rate or rhythm management, and risk factor modification to reduce complications. Companies marketing AF screening devices should report the accuracy and performance of their products in high- and low-risk populations and avoid claims about clinical outcomes unless improvement is demonstrated in randomized clinical trials. Generally, the diagnostic yield of AF screening increases with the number, duration, and temporal dispersion of screening sessions, but the prognostic importance may be less than for AF detected by single-time point screening, which is largely permanent, persistent, or high-burden paroxysmal AF. Consumer-initiated ECG recordings suggesting possible AF always require confirmation by a health care professional experienced in ECG reading, whereas suspicion of AF on the basis of photoplethysmography must be confirmed with an ECG. Consumer-led AF screening is unlikely to be cost-effective for stroke prevention in the predominantly young, early adopters of this technology. Studies in older people at higher stroke risk are required to demonstrate both effectiveness and cost-effectiveness. The direct interaction between companies and consumers creates new regulatory gaps in relation to data privacy and the registration of consumer apps and devices. Although several barriers for optimal use of consumer-led screening exist, results of large, ongoing trials, powered to detect clinical outcomes, are required before health care professionals should support widespread adoption of consumer-led AF screening.

Atrial Fibrillation and Dementia: A Report From the AF-SCREEN International Collaboration.

Growing evidence suggests a consistent association between atrial fibrillation (AF) and cognitive impairment and dementia that is independent of clinical stroke. This report from the AF-SCREEN International Collaboration summarizes the evidence linking AF to cognitive impairment and dementia. It provides guidance on the investigation and management of dementia in patients with AF on the basis of best available evidence. The document also addresses suspected pathophysiologic mechanisms and identifies knowledge gaps for future research. Whereas AF and dementia share numerous risk factors, the association appears to be independent of these variables. Nevertheless, the evidence remains inconclusive regarding a direct causal effect. Several pathophysiologic mechanisms have been proposed, some of which are potentially amenable to early intervention, including cerebral microinfarction, AF-related cerebral hypoperfusion, inflammation, microhemorrhage, brain atrophy, and systemic atherosclerotic vascular disease. The mitigating role of oral anticoagulation in specific subgroups (eg, low stroke risk, short duration or silent AF, after successful AF ablation, or atrial cardiopathy) and the effect of rhythm versus rate control strategies remain unknown. Likewise, screening for AF (in cognitively normal or cognitively impaired patients) and screening for cognitive impairment in patients with AF are debated. The pathophysiology of dementia and therapeutic strategies to reduce cognitive impairment warrant further investigation in individuals with AF. Cognition should be evaluated in future AF studies and integrated with patient-specific outcome priorities and patient preferences. Further large-scale prospective studies and randomized trials are needed to establish whether AF is a risk factor for cognitive impairment, to investigate strategies to prevent dementia, and to determine whether screening for unknown AF followed by targeted therapy might prevent or reduce cognitive impairment and dementia.

Smartphone Imaging Flow Cytometry for High-Throughput Single-Cell Analysis.

We present a portable imaging flow cytometer comprising a smartphone, a small-footprint optical framework, and a PDMS-based microfluidic device. Flow cytometric analysis is performed in a sheathless manner via elasto-inertial focusing with a custom-written Android program, integrating a graphical user interface (GUI) that provides a high degree of user control over image acquisition. The proposed system offers two different operational modes. First, "post-processing" mode enables particle/cell sizing at throughputs of up to 67 000 particles/s. Alternatively, "real-time" mode allows for integrated cell/particle classification with machine learning at throughputs of 100 particles/s. To showcase the efficacy of our platform, polystyrene particles are accurately enumerated within heterogeneous populations using the post-processing mode. In real-time mode, an open-source machine learning algorithm is deployed within a custom-developed Android application to classify samples containing cells of similar size but with different morphologies. The flow cytometer can extract high-resolution bright-field images with a spatial resolution <700 nm using the developed machine learning-based algorithm, achieving classification accuracies of 97% and 93% for Jurkat and EL4 cells, respectively. Our results confirm that the smartphone imaging flow cytometer (sIFC) is capable of both enumerating single particles in flow and identifying morphological features with high resolution and minimal hardware.

Same-day discharge for left atrial appendage occlusion procedure: A systematic review and meta-analysis.

INTRODUCTION: Most patients undergoing a left atrial appendage occlusion (LAAO) procedure are admitted for overnight observation. A same-day discharge strategy offers the opportunity to improve resource utilization without compromising patient safety. We compared the patient safety outcomes and post-discharge complications between same-day discharge versus hospital admission (HA) (>1 day) in patients undergoing LAAO procedure. METHODS: A systematic search of MEDLINE and Embase was conducted. Outcomes of interest included peri-procedural complications, re-admissions, discharge complications including major bleeding and vascular complications, ischemic stroke, all-cause mortality, and peri-device leak >5 mm. Mantel-Haenszel risk ratios (RRs) with 95% CIs were calculated. RESULTS: A total of seven observational studies met the inclusion criteria. There was no statistically significant difference between same-day discharge versus HA regarding readmission (RR: 0.61; 95% confidence interval [CI]: [0.29-1.31]; p = .21), ischemic stroke after discharge (RR: 1.16; 95% CI: [0.49-2.73]), peri-device leak >5 mm (RR: 1.27; 95% CI: [0.42-3.85], and all-cause mortality (RR: 0.60; 95% CI: [0.36-1.02]). The same-day discharge study group had significantly lower major bleeding or vascular complications (RR: 0.71; 95% CI: [0.54-0.94]). CONCLUSIONS: This meta-analysis of seven observational studies showed no significant difference in patient safety outcomes and post-discharge complications between same-day discharge versus HA. These findings provide a solid basis to perform a randomized control trial to eliminate any potential confounders.

The effect of weight loss on recurrence of atrial fibrillation after catheter ablation: A systematic review and meta-analysis.

BACKGROUND: Obesity is associated with an increased risk of developing recurrent atrial fibrillation (AF) after catheter ablation (CA). However, the current data on weight loss interventions show inconsistent results in preventing the recurrence of AF after CA. METHODS: We conducted a systematic search in MEDLINE and EMBASE to identify studies that reported the outcome of recurrence of AF after CA in obese patients undergoing weight interventions. The subgroup analysis included: (1) Weight loss versus no weight loss, (2) >10% weight loss versus <10% weight loss, (3) <10% weight loss versus no weight loss, (4) Follow-up <12 months, and (5) Follow-up >12 months after CA. Mantel-Haenszel risk ratios with a 95% confidence interval (CI) were calculated using a random effects model and for heterogeneity, I2 statistics were reported. RESULTS: A total of 10 studies (one randomized controlled trial and nine observational studies) comprising 1851 patients were included. The recurrence of AF was numerically reduced in the weight loss group (34.5%) versus no weight loss group (58.2%), but no statistically significant difference was observed (risk ratio [RR] = 0.76; 95% CI: 0.49-1.18, p = .22). However, there was a statistically significant reduction in recurrence of AF with weight loss versus no weight loss at follow-up >12 months after CA (RR = 0.47; 95% CI: 0.32-0.68, p < .0001). At follow-up >12 months after CA, both >10% weight loss versus <10% weight loss (RR = 0.49; 95% CI: 0.31-0.80, p = .004) and <10% weight loss versus no weight loss (RR = 0.39; 95% CI: 0.31-0.49, p < .00001) were associated with a statistically significant reduction in recurrent AF. CONCLUSION: In patients with AF undergoing CA, weight loss is associated with reducing recurrent AF at > 12 months after ablation and these benefits are consistently seen with both >10% and <10% weight loss. The benefits of weight loss in preventing recurrent AF after CA should be examined in larger studies with extended follow-up duration.

Association of atrial fibrillation and outcomes in patients undergoing bone marrow transplantation.

AIMS: Haematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for several malignant and non-malignant haematologic conditions. Patients undergoing HSCT are at an increased risk of developing atrial fibrillation (AF). We hypothesized that a diagnosis of AF would be associated with poor outcomes in patients undergoing HSCT. METHODS AND RESULTS: The National Inpatient Sample (2016-19) was queried with ICD-10 codes to identify patients aged >50 years undergoing HSCT. Clinical outcomes were compared between patients with and without AF. A multivariable regression model adjusting for demographics and comorbidities was used to calculate the adjusted odds ratio (aOR) and regression coefficients with corresponding 95% confidence intervals and P-values. A total of 50 570 weighted hospitalizations for HSCT were identified, out of which 5820 (11.5%) had AF. Atrial fibrillation was found to be independently associated with higher inpatient mortality (aOR 2.75; 1.9-3.98; P < 0.001), cardiac arrest (aOR 2.86; 1.55-5.26; P = 0.001), acute kidney injury (aOR 1.89; 1.6-2.23; P < 0.001), acute heart failure exacerbation (aOR 5.01; 3.54-7.1; P < 0.001), cardiogenic shock (aOR 7.73; 3.17-18.8; P < 0.001), and acute respiratory failure (aOR 3.24; 2.56-4.1; P < 0.001) as well as higher mean length of stay (LOS) (+2.67; 1.79-3.55; P < 0.001) and cost of care (+67 529; 36 630-98 427; P < 0.001). CONCLUSION: Among patients undergoing HSCT, AF was independently associated with poor in-hospital outcomes, higher LOS, and cost of care.

Safety and efficacy of colchicine for the prevention of post-operative atrial fibrillation in patients undergoing cardiac surgery: a meta-analysis of randomized controlled trials.

BACKGROUND AND AIMS: Colchicine is an anti-inflammatory drug that may prevent post-operative atrial fibrillation (POAF). The effect of this drug has been inconsistently shown in previous clinical trials. We aimed to compare the efficacy and safety of colchicine vs. placebo to prevent POAF in patients undergoing cardiac surgery. METHODS AND RESULTS: A systematic search of EMBASE, MEDLINE, SCOPUS, ClinicalTrials.gov, and the Cochrane Library for randomized controlled trials (RCTs) was conducted from inception till April 2023. The primary outcome was the incidence of POAF after any cardiac surgery. The secondary outcome was the rate of drug discontinuation due to adverse events and adverse gastrointestinal events. Risk ratios (RR) were reported using the Mantel Haenszel method. A total of eight RCTs comprising 1885 patients were included. There was a statistically significant lower risk of developing POAF with colchicine vs. placebo (RR: 0.70; 95% CI: 0.59-0.82; P < 0.01, I2 = 0%), and this effect persisted across different subgroups. There was a significantly higher risk of adverse gastrointestinal events (RR: 2.20; 95% CI: 1.38-3.51; P < 0.01, I2 = 55%) with no difference in the risk of drug discontinuation in patients receiving colchicine vs. placebo (RR: 1.33; 95% CI: 0.93-1.89; P = 0.11, I2 = 0%). CONCLUSION: This meta-analysis of eight RCTs shows that colchicine is effective at preventing POAF, with a significantly higher risk of adverse gastrointestinal events but no difference in the rate of drug discontinuation. Future studies are required to define the optimal duration and dose of colchicine for the prevention of POAF.

Psychosocial risk factors and outcomes in patients undergoing catheter ablation for atrial fibrillation.

The association of psychosocial risk factors with cardiovascular disease is well-established, and there is a growing recognition of their influence on atrial fibrillation (AF) . A recent National Heart, Lung, and Blood Institute workshop called for transforming AF research to integrate social determinants of health. There is limited data examining the impact of psychosocial risk factors (PSRFs) on outcomes in patients with an established diagnosis of AF. Catheter ablation for AF has been shown to improve arrhythmia burden and quality of life compared with medical treatment alone. It is unknown how PSRFs affect clinical outcomes in patients undergoing AF ablation. It is important to understand this relationship, especially given the increasing adoption of catheter ablation in clinical practice.

Transforming Nanomaterial Synthesis with Flow Chemistry.

Microfluidic methods for the synthesis of nanomaterials allow the generation of high-quality products with outstanding structural, electronic and optical properties. At a fundamental level, this is engendered by the ability to control both heat and mass transfer in a rapid and precise manner, but also by the facile integration of in-line characterization tools and machine learning algorithms. Such integrated platforms provide for exquisite control over material properties during synthesis, accelerate the optimization of electronic and optical properties and bestow new insights into the optoelectronic properties of nanomaterials. Herein, we present a brief perspective on the role that microfluidic technologies can play in nanomaterial synthesis, with a particular focus on recent studies that incorporate in-line optical characterization and machine learning. We also consider the importance and challenges associated with integrating additional functional components within experimental workflows and the upscaling of microfluidic platforms for production of industrial-scale quantities of nanomaterials.

Quantifying Antibody Binding Kinetics on Fixed Cells and Tissues via Fluorescence Lifetime Imaging.

We present a method for monitoring spatially localized antigen-antibody binding events on physiologically relevant substrates (cell and tissue sections) using fluorescence lifetime imaging. Specifically, we use the difference between the fluorescence decay times of fluorescently tagged antibodies in free solution and in the bound state to track the bound fraction over time and hence deduce the binding kinetics. We make use of a microfluidic probe format to minimize the mass transport effects and localize the analysis to specific regions of interest on the biological substrates. This enables measurement of binding constants (kon) on surface-bound antigens and on cell blocks using model biomarkers. Finally, we directly measure p53 kinetics with differential biomarker expression in ovarian cancer tissue sections, observing that the degree of expression corresponds to the changes in kon, with values of 3.27-3.50 × 103 M-1 s-1 for high biomarker expression and 2.27-2.79 × 103 M-1 s-1 for low biomarker expression.

Droplet-Based Microfluidic Temperature-Jump Platform for the Rapid Assessment of Biomolecular Kinetics.

Protein folding, unfolding, and aggregation are important in a variety of biological processes and intimately linked to "protein misfolding diseases". The ability to perform experiments at different temperatures allows the extraction of important information regarding the kinetics and thermodynamics of such processes. Unfortunately, conventional stopped-flow methods are difficult to implement, generate limited information, and involve complex sample handling. To address this issue, we present a temperature-controlled droplet-based microfluidic platform that allows measurement of reaction kinetics on millisecond to second timescales and at temperatures between ambient and 90 °C. The utility of the microfluidic platform for measuring fast biomolecular kinetics at high temperatures is showcased through the investigation of the unfolding kinetics of haloalkane dehalogenases and the elongation of fibrils composed of the amyloid ß peptide associated with Alzheimer's disease. In addition, a deep-ultraviolet (UV) fluorescence microscope was developed for the on-chip recording of protein intrinsic fluorescence spectrum originating from aromatic amino acid residues. We envision that the developed optofluidic platform will find wide applicability in the analysis of biological processes, such as protein refolding and phase separation.

Droplet Microfluidics for the Label-Free Extraction of Complete Phase Diagrams and Kinetics of Liquid-Liquid Phase Separation in Finite Volumes.

Liquid-liquid phase separation of polymer and protein solutions is central in many areas of biology and material sciences. Here, an experimental and theoretical framework is provided to investigate the thermodynamics and kinetics of liquid-liquid phase separation in volumes comparable to cells. The strategy leverages droplet microfluidics to accurately measure the volume of the dense phase generated by liquid-liquid phase separation of solutions confined in micro-sized compartments. It is shown that the measurement of the volume fraction of the dense phase at different temperatures allows the evaluation of the binodal lines that determine the coexistence region of the two phases in the temperature-concentration phase diagram. By applying a thermodynamic model of phase separation in finite volumes, it is further shown that the platform can predict and validate kinetic barriers associated with the formation of a dense droplet in a parent dilute phase, therefore connecting thermodynamics and kinetics of liquid-liquid phase separation.

Hybrid Microfluidic Device for High Throughput Isolation of Cells Using Aptamer Functionalized Diatom Frustules.

Circulating tumor cells (CTCs), secreted from primary and metastatic malignancies, hold a wealth of essential diagnostic and prognostic data for multiple cancers. Significantly, the information contained within these cells may hold the key to understanding cancer metastasis, both individually and fundamentally. Accordingly, developing ways to identify, isolate and interrogate CTCs plays an essential role in modern cancer research. Unfortunately, CTCs are typically present in the blood in vanishingly low titers and mixed with other blood components, making their isolation and analysis extremely challenging. Herein, we report the design, fabrication and optimization of a microfluidic device capable of automatically isolating CTCs from whole blood. This is achieved in two steps, via the passive viscoelastic separation of CTCs and white blood cells (WBCs) from red blood cells (RBCs), and subsequent active magnetophoretic separation of CTCs from WBCs. We detail the specific geometries required to balance the elastic and inertial forces required for successful passive separation of RBCs, and the use of computational fluid dynamics (CFD) to optimize active magnetophoretic separation. We subsequently describe the use of magnetic biosilica frustules, extracted from Chaetoceros sp. diatoms, to fluorescently tag CTCs and facilitate magnetic isolation. Finally, we use our microfluidic platform to separate HepG2-derived CTCs from whole blood, demonstrating exceptional CTC recovery (94.6%) and purity (89.7%).

Broad-Band Spectrum, High-Sensitivity Absorbance Spectroscopy in Picoliter Volumes.

Picoliter-volume droplets within segmented flows can be probed in a rapid and efficient manner using optical detection methods. To date, however, most detection schemes for droplet content analysis have relied on the use of time-integrated fluorescence measurements. Despite its undoubted utility, the implementation of absorbance-based detectors is particularly challenging due to the reduced optical path lengths that are characteristic of microfluidic systems and deleterious scattering at droplet-oil interfaces. Unsurprisingly, efforts to develop sensitive absorbance-based detection schemes for the interrogation of rapidly moving droplets have primarily focused on ensuring adequate analytical sensitivity and, to date, have been exclusively limited to single-wavelength measurements. To address this limitation, and expand the information content associated with absorbance measurements on-chip, we herein describe a detection scheme for the extraction of broad-band absorbance spectra from pL-volume droplets with high sensitivity. The combination of a confocal optical system (that confines incident light to a reduced detection volume) and a postprocessing algorithm (that effectively removes the contribution of the carrier oil from the extracted spectra) engenders significant improvements in signal-to-noise ratios. Our system is initially calibrated by acquiring absorbance spectra from aqueous solutions of fluorescein isothiocyanate. These measurements confirm both excellent linearity over the studied range (from 0 to 100 µM) and a concentration limit of detection of 800 nM. The methodology is then used to monitor the salt-induced aggregation of gold nanoparticles with millisecond time resolution. This approach for small-volume absorbance spectroscopy allows for both high-throughput and high-information content measurements in subnanoliter volumes and will be highly desirable in a wide variety of bioanalytical applications where sensitivity and throughput are priorities.