Asynchronous magnetic bead rotation microviscometer for rapid, sensitive, and label-free studies of bacterial growth and drug sensitivity.

The long turnaround time in antimicrobial susceptibility testing (AST) endangers patients and encourages the administration of wide spectrum antibiotics, thus resulting in alarming increases of multidrug resistant pathogens. A method for faster detection of bacterial proliferation presents one avenue toward addressing this global concern. We report on a label-free asynchronous magnetic bead rotation (AMBR) based viscometry method that rapidly detects bacterial growth and determines drug sensitivity by measuring changes in the suspension's viscosity. With this platform, we observed the growth of a uropathogenic Escherichia coli isolate, with an initial concentration of 50 cells per drop, within 20 min; in addition, we determined the gentamicin minimum inhibitory concentration (MIC) of the E. coli isolate within 100 min. We thus demonstrated a label-free, microviscometer platform that can measure bacterial growth and drug susceptibility more rapidly, with lower initial bacterial counts than existing commercial systems, and potentially with any microbial strains.

Self-assembled magnetic bead biosensor for measuring bacterial growth and antimicrobial susceptibility testing.

Bacterial antibiotic resistance is one of the major concerns of modern healthcare worldwide, and the development of rapid, growth-based, antimicrobial susceptibility tests is key for addressing it. The cover image shows a self-assembled asynchronous magnetic bead rotation (AMBR) biosensor developed for rapid detection of bacterial growth. Using the biosensors, the minimum inhibitory concentration of a clinical E. coli isolate can be measured within two hours, where currently tests take 6-24 hours. A 16-well prototype is also constructed for simple and robust observation of the self-assembled AMBR biosensors.

Characterization of vascular injury responses to stent insertion in an ex-vivo arterial perfusion model.

PURPOSE: To develop an ex-vivo arterial perfusion model to evaluate vascular responses to bare metal stents (BMS) and drug-eluting stents (DES) in porcine carotid arteries. MATERIALS AND METHODS: Porcine carotid arteries with BMS or DES were cultured under hemodynamic stimuli for 24 hours and 72 hours. Vascular responses of arteries with stents were assessed by cellular functionality and gene expression and compared with a noninjured (NI) control group at each time point. Cellular functionality was confirmed with sequential dosing of norepinephrine (NE), acetylcholine (ACH), and sodium nitroprusside (SNP). QuantiGene (Panomics, Fremont, California) branched DNA (bDNA) assay was used to evaluate gene expression of endothelial cell (EC) and smooth muscle cell (SMC) biomarkers and compare it with responses of in-vivo arteries with stents. Bromodeoxyuridine (BrDU) stain was also used to detect cellular proliferation in the ex-vivo arteries with stents. RESULTS: EC relaxation and SMC contraction in response to vasoactivators indicated the arteries remained viable and functional for at least 72 hours in culture. SMC-dependent contractility and EC-dependent relaxation were lower in arteries with stents compared with NI arteries. Greater SMC proliferation was observed in BMS arteries compared with DES arteries. Cellular proliferation, EC function, and SMC marker expression at acute time points were similar between both models suggesting that the ex-vivo arterial model can provide comparative predictions of stent injury in vivo. CONCLUSIONS: The ex-vivo arterial perfusion model can be used as a quick and less costly (than current in-vivo and some in-vitro perfusion testing models) approach for evaluating the vascular responses to various stent design parameters (eg, strut thickness, strut width).

Microfluidic chemical analysis systems.

The field of microfluidics has exploded in the past decade, particularly in the area of chemical and biochemical analysis systems. Borrowing technology from the solid-state electronics industry and the production of microprocessor chips, researchers working with glass, silicon, and polymer substrates have fabricated macroscale laboratory components in miniaturized formats. These devices pump nanoliter volumes of liquid through micrometer-scale channels and perform complex chemical reactions and separations. The detection of reaction products is typically done fluorescently with off-chip optical components, and the analysis time from start to finish can be significantly shorter than that of conventional techniques. In this review we describe these microfluidic analysis systems, from the original continuous flow systems relying on electroosmotic pumping for liquid motion to the large diversity of microarray chips currently in use to the newer droplet-based devices and segmented flow systems. Although not currently widespread, microfluidic systems have the potential to become ubiquitous.

Asynchronous magnetic bead rotation (AMBR) biosensor in microfluidic droplets for rapid bacterial growth and susceptibility measurements.

Inappropriate antibiotic use is a major factor contributing to the emergence and spread of antimicrobial resistance. The long turnaround time (over 24 hours) required for clinical antimicrobial susceptibility testing (AST) often results in patients being prescribed empiric therapies, which may be inadequate, inappropriate, or overly broad-spectrum. A reduction in the AST time may enable more appropriate therapies to be prescribed earlier. Here we report on a new diagnostic asynchronous magnetic bead rotation (AMBR) biosensor droplet microfluidic platform that enables single cell and small cell population growth measurements for applications aimed at rapid AST. We demonstrate the ability to rapidly measure bacterial growth, susceptibility, and the minimum inhibitory concentration (MIC) of a small uropathogenic Escherichia coli population that was confined in microfluidic droplets and exposed to concentrations above and below the MIC of gentamicin. Growth was observed below the MIC, and no growth was observed above the MIC. A 52% change in the sensor signal (i.e. rotational period) was observed within 15 minutes, thus allowing AST measurements to be performed potentially within minutes.

Monitoring the growth and drug susceptibility of individual bacteria using asynchronous magnetic bead rotation sensors.

Continuous growth of individual bacteria has been previously studied by direct observation using optical imaging. However, optical microscopy studies are inherently diffraction limited and limited in the number of individual cells that can be continuously monitored. Here we report on the use of the asynchronous magnetic bead rotation (AMBR) sensor, which is not diffraction limited. The AMBR sensor allows for the measurement of nanoscale growth dynamics of individual bacterial cells, over multiple generations. This torque-based magnetic bead sensor monitors variations in drag caused by the attachment and growth of a single bacterial cell. In this manner, we observed the growth and division of individual Escherichia coli, with 80-nm sensitivity to the cell length. Over the life cycle of a cell, we observed up to a 300% increase in the rotational period of the biosensor due to increased cell volume. In addition, we observed single bacterial cell growth response to antibiotics. This work demonstrates the non-microscopy limited AMBR biosensor for monitoring individual cell growth dynamics, including cell elongation, generation time, lag time, and division, as well as their sensitivity to antibiotics.

Vascular response to coronary artery stenting in mature and juvenile swine.

PURPOSE: The objective of this study was to investigate potential differences in vascular response to stenting of coronary arteries with bare metal (BMS) and drug-eluting (DES) stents in juvenile vs. mature swine. METHODS AND MATERIALS: Twenty-one mature (> 3 years) and 22 juvenile (6-9 months) Yucatan swine were implanted with 3 × 12-mm XIENCE V DES and ML VISION BMS in coronary arteries. After 7 and 28 days, vessels were analyzed using light microscopy (n = 5-7) and confocal and scanning electron microscopy (n = 5-10). Messenger RNA expression levels of inflammatory and endothelial gene markers were tested from stented tissue at 7 and 28 days (n = 3). A 2 × 2 analysis of variance followed by t tests compared treatment and/or age effects. RESULTS: No age differences in neointimal area and percentage stenosis were measured. Juvenile swine exhibited increased fibrin scores compared to mature swine (2.6 ± 0.5 vs. 2.2 ± 0.5, P < .05) at 7 days, with no age-related difference at 28 days. At 7 days, significant increases in para-strut inflammation (P < .01) and in VCAM-1, ICAM-1, CD40 and MCP-1 gene expression (P < .05) were observed in mature swine, but differences were largely resolved by 28 days. DES exhibited less endothelial coverage than BMS at 7 days, but this difference was abrogated by 28 days, with no difference between age groups. CONCLUSIONS: Our results indicate that mature swine exhibited an increased foreign body response compared to mature swine at 7 and 28 days following stenting that may indicate marginal delays in resolution of foreign body response in aged populations. These differences are unlikely to affect methodologies for preclinical stent safety evaluations.

High frequency asynchronous magnetic bead rotation for improved biosensors.

Biosensors with increasingly high sensitivity are crucial for probing small scale properties. The asynchronous magnetic bead rotation (AMBR) sensor is an emerging sensor platform, based on magnetically actuated rotation. Here the frequency dependence of the AMBR sensor's sensitivity is investigated. An asynchronous rotation frequency of 145 Hz is achieved. This increased frequency will allow for a calculated detection limit of as little as a 59 nm change in bead diameter, which is a dramatic improvement over previous AMBR sensors and further enables physical and biomedical applications.