Search

Identifying nonhormonal contraceptives will have profound impacts on avoiding side effects of hormonal birth control methods, minimizing pregnancy complications and infant mortality rates, and promoting family planning. However, phenotypic screening of contraceptives is challenging due to the diverse procedures associated with oocyte culture, biochemical assays, and molecular imaging. This study reports a multifunctional microfluidic platform comprising reconfigurable building blocks and interfaces to implement various cell-based drug screening protocols. This versatile platform has three major layers. The top layer consists of interchangeable 3D microfluidic building blocks (e.g., branching microchannels, chemical gradient generators, pumpless flow controllers, and emulsion generators) or an open interface. The middle layer incorporates a multiwell array with embedded membrane filters for live cell culture, medium exchange, enzymatic cumulus cell removal, washing, and fluorescence staining. The bottom layer is also reconfigurable for waste collection, oocyte culture, plate reader measurement, and high-resolution microscopy. We demonstrate an 8 by 16 (128 wells) system for performing the cumulus–oocyte complex (COC) expansion and oocyte maturation assays for screening nonhormonal contraceptives. The microfluidic building block platform is scalable and can be reconfigured for a variety of drug screening applications in the future. [ABSTRACT FROM AUTHOR]

Double-stranded (ds) biosensors are homogeneous oligonucleotide probes for detection of nucleic acid sequences in biochemical assays and live cell imaging. Locked nucleic acid (LNA) modification can be incorporated in the biosensors to enhance the binding affinity, specificity, and resistance to nuclease degradation. However, LNA monomers in the quencher sequence can also prevent the target-fluorophore probe binding, which reduces the signal of the dsLNA biosensor. This study investigates the influence of LNA modification on dsLNA biosensors by altering the position and amount of LNA monomers present in the quencher sequence. We characterize the fluorophore–quencher interaction, target detection, and specificity of the biosensor in free solution and evaluate the performance of the dsLNA biosensor in 2D monolayers and 3D spheroids. The data indicate that a large amount of LNA monomers in the quencher sequence can enhance the specificity of the biosensor, but prevents effective target binding. Together, our results provide guidelines for improving the performance of dsLNA biosensors in nucleic acid detection and gene expression analysis in live cells. [ABSTRACT FROM AUTHOR]

We report an on-chip platform for low-intensity pulsed ultrasound (LIPUS) stimulation of cells directly cultured on a biocompatible surface of a transparent ultrasound transducer (TUT) fabricated using lithium niobate. The high light transmittance (>80%) and compact size (3 mm × 3 mm × 2 mm) of TUTs allowed easy integration with powerful optical microscopy techniques with no additional acoustic coupling and risk for contamination. TUTs were excited with varying acoustic excitation parameters (voltage amplitude and duty cycle) and resulting live cell calcium signaling was simultaneously imaged using time-lapse confocal microscopy, while the temperature change was measured by a thermocouple. Quantitative single-cell fluorescence analysis revealed the dynamic calcium signaling responses and together with the temperature measurements elucidated the optimal stimulation parameters for non-thermal and thermal effects. The fluorescence change profile was distinct from the recorded temperature change (<1 degree Celsius) profile under LIPUS treatment conditions. Cell dead assay results confirmed cells remain viable after the LIPUS treatment. These results confirmed that the TUT platform enables controllable, safe, high-throughput, and uniform mechanical stimulation of all plated cells. The on-chip LIPUS stimulation using TUTs has the potential to attract several in vitro and in vivo biomedical applications such as controlling stem cell differentiation and proliferation, studying biomechanical properties of cancer cells, and gaining fundamental insights into mechanotransduction pathways when integrated with state-of-the-art high-speed and high-resolution microscopy techniques. [ABSTRACT FROM AUTHOR]

Single-molecule and single-cell analysis techniques have opened new opportunities for characterizing and analyzing heterogeneity within biological samples. These detection methods are often referred to as digital assays because the biological sample is partitioned into many small compartments and each compartment contains a discrete number of targets (e.g. cells). Using digital assays, researchers can precisely detect and quantify individual targets, and this capability has made digital techniques the basis for many modern bioanalytical tools (including digital PCR, single cell RNA sequencing, and digital ELISA). However, digital assays are dominated by optical analysis systems that typically utilize microscopy to analyze partitioned samples. The utility of digital assays may be dramatically enhanced by implementing cost-efficient and portable electrical detection capabilities. Herein, we describe a digital electrical impedance sensing platform that enables direct multiplexed measurement of single cell bacterial cells. We outline our solutions to the challenge of multiplexing impedance sensing across many culture compartments and demonstrate the potential for rapidly differentiating antimicrobial resistant versus susceptible strains of bacteria. [ABSTRACT FROM AUTHOR]

Point-of-care (POC) diagnostics for infectious diseases have the potential to improve patient care and antibiotic stewardship. Nucleic acid hybridization is at the core of many amplification-free molecular diagnostics and detection probe configuration is key to diagnostic performance. Modified nucleic acids such as peptide nucleic acid (PNA) offer advantages compared to conventional DNA probes allowing for faster hybridization, better stability and minimal sample preparation for direct detection of pathogens. Probes with tethered fluorophore and quencher allow for solution-based assays and eliminate the need for washing steps thereby facilitating integration into microfluidic devices. Here, we compared the sensitivity and specificity of double stranded PNA probes (dsPNA) and PNA molecular beacons targeting E. coli and P. aeruginosa for direct detection of bacterial pathogens. In bulk fluid assays, the dsPNAs had an overall higher fluorescent signal and better sensitivity and specificity than the PNA beacons for pathogen detection. We further designed and tested an expanded panel of dsPNA probes for detection of a wide variety of pathogenic bacteria including probes for universal detection of eubacteria, Enterobacteriaceae family, and P. mirablis. To confirm that the advantage translated to other assay types we compared the PNA beacon and dsPNA in a prototype droplet microfluidic device. Beyond the bulk fluid assay and droplet devices, use of dsPNA probes may be advantageous in a wide variety of assays that employ homogenous nucleic acid hybridization. [ABSTRACT FROM AUTHOR]

Amperometric biosensors are widely applied for rapid biomarker detection in physiological and environmental samples. The dynamics and linearity of the current signal, however, are only partially understood. This study investigates the diffusion–reaction kinetics of amperometric biosensing using a self-assembled monolayer (SAM) based biosensor for bacterial 16S rRNA. A numerical model is developed to optimize the chamber dimensions and elucidate the concentration dependences of the biosensor. The results revealed that depletion of substrates associated with the chamber dimension can limit the current signal in a target concentration dependent manner. This study provides practical guidelines in the design and interpretation of microfluidic amperometric biosensors for biochemical applications. [ABSTRACT FROM AUTHOR]

Double-stranded probes are homogeneous biosensors for rapid detection of specific nucleotide sequences. These double-stranded probes have been applied in various molecular sensing applications, such as real-time polymerase chain reaction and detection of bacterial 16S rRNA. In this study, we present the design and optimization of double-stranded probes for single-cell gene expression analysis in living cells. With alternating DNA/LNA monomers for optimizing the stability and specificity, we show that the probe is stable in living cells for over 72 hours post-transfection and is capable of detecting changes in gene expression induced by external stimuli. The probes can be delivered to a large number of cells simultaneously by cationic liposomal transfection or to individual cells selectively by photothermal delivery. We also demonstrate that the probe quantifies intracellular mRNA in living cells through the use of an equilibrium analysis. With its effectiveness and performance, the double-stranded probe represents a broadly applicable approach for large-scale single-cell gene expression analysis toward numerous biomedical applications, such as systems biology, cancer, and drug screening. [ABSTRACT FROM AUTHOR]

To develop a portable point-of-care system based on biosensors for common infectious diseases such as urinary tract infection, the sensing process needs to be implemented within an enclosed fluidic system. On chip sample preparation of clinical samples remains a significant obstacle to achieving robust sensor performance. Herein AC electrokinetics is applied in an electrochemical biosensor cassette to enhance molecular convection and hybridization efficiency through electrokinetics induced fluid motion and Joule heating induced temperature elevation. Using E. coli as an exemplary pathogen, we determined the optimal electrokinetic parameters for detecting bacterial 16S rRNA in the biosensor cassette based on the current output, signal-to-noise ratio, and limit of detection. In addition, a panel of six probe sets targeting common uropathogenic bacteria was demonstrated. The optimized parameters were also validated using patient-derived clinical urine samples. The effectiveness of electrokinetics for on chip sample preparation will facilitate the implementation of point-of-care diagnosis of urinary tract infection in the future. [ABSTRACT FROM AUTHOR]

This study reports the hybridization kinetics of double-stranded DNA probes for rapid molecular analysis. Molecular binding schemes based on double-stranded DNA probes have been developed for quantitative detection of various biomolecules, such as nucleic acids and DNA binding proteins recently. The thermodynamic competition between the target and the competitor in binding to the probe provides a highly specific mechanism for molecular detection. The kinetics of the double-stranded DNA probe, on the other hand, represent another key aspect toward its general applicability for a wide set of biomedical applications. Herein we report a systematic investigation of the kinetics of double-stranded DNA probes. The signal-to-background ratio and assay time of the double-stranded DNA probes are optimized at a high ionic strength (over 100 mM NaCl). Both the donor probe and the quencher probe sequences are shown to be important in the hybridization kinetics. A long sticky end of the probe is able to dramatically accelerate the kinetics of the assay. To provide a quantitative description of the kinetics, a two-stage binding model is developed to describe the major features of the kinetics of the assay. The sensitivity of the kinetic model and the dominant affinity constants are studied. The study provides a general guideline for the design of the probes for reducing the total assay time. With an appropriate design of the probes, the assay can be finished within minutes at room temperature. [ABSTRACT FROM AUTHOR]

Active manipulation of cells, such as trapping, focusing, and isolation, is essential for various bioanalytical applications. Herein, we report a hybrid electrokinetic technique for manipulating mammalian cells in physiological fluids. This technique applies a combination of negative dielectrophoretic force and hydrodynamic drag force induced by electrohydrodynamics, which is effective in conductive biological fluids. With a three-electrode configuration, the stable equilibrium positions of cells can be adjusted for separation and focusing applications. Cancer cells and white blood cells can be positioned and isolated into specific locations in the microchannel under both static and dynamic flow conditions. To investigate the sensitivity of the hybrid electrokinetic process, AC voltage, frequency, and bias dependences of the cell velocity were studied systematically. The applicability of the hybrid electrokinetic technique for manipulating cells in physiological samples is demonstrated by continuous focusing of human breast adenocarcinoma spiked in urine, buffy coats, and processed blood samples with 98% capture efficiency.

This study reports a hybrid electrokinetic technique for label-free manipulation of pathogenic bacteria in biological samples toward medical diagnostic applications. While most electrokinetic techniques only function in low-conductivity buffers, hybrid electrokinetics enables effective operation in high-conductivity samples, such as physiological fluids (∼1 S m(-1)). The hybrid electrokinetic technique combines short-range electrophoresis and dielectrophoresis, and long-range AC electrothermal flow to improve its effectiveness. The major technical hurdle of electrode instability for manipulating high conductivity samples is tackled by using a Ti-Au-Ti sandwich electrode and a 3-parallel-electrode configuration is designed for continuous isolation of bacteria. The device operates directly with biological samples including urine and buffy coats. We show that pathogenic bacteria and biowarfare agents can be concentrated for over 3 orders of magnitude using hybrid electrokinetics., (© The Royal Society of Chemistry 2011)

This paper reports a simple, rapid molecular binding scheme for the detection and quantification of the transcription factor nuclear factor kappa B and other DNA binding proteins without any separation or immobilization step.

This paper reports the design and optimization of a double-stranded molecular probe for homogeneous detection of specific nucleotide sequences. The probes are labeled with either a fluorophore or a quencher such that the probe hybridization brings the two labels into close proximity, and this diminishes the fluorescence signal in the absence of a target. In the presence of a target, the fluorophore probe is thermodynamically driven to unzip from its hybridized form and bind with the target. An equilibrium analysis, which successfully describes all the major features of the assay without any fitting parameter, is performed to generalize the design of the probe. Several key parameters affecting the performance of the assay are examined. We show that the dynamic range and the signal-to-noise ratio of the assay can be optimized by the probe concentration, the quencher-to-fluorophore ratio, and the probe strand sequence. By proper design of the sequence, the probe discriminates single nucleotide mismatches in a single step without any separation step or measurement of melting profile.