Highly Sensitive, Portable Detection System for Multiplex Chemiluminescence Analysis

Chemiluminescence (CL) has emerged as a critical tool for the sensing and quantification of various bioanalytes in virtually all clinical fields. However, the rapid nature of many CL reactions raises challenges for typical low-cost optical sensors such as cameras to achieve accurate and sensitive detection. Meanwhile, classic sensors such as photomultiplier tubes are highly sensitive but lack spatial multiplexing capabilities and are generally not suited for point-of-care applications outside a standard laboratory setting. To address this issue, in this paper, a miniaturized and versatile silicon-photomultiplier-based fiber-integrated CL device (SFCD) was designed for sensitive multiplex CL detection. The SFCD comprises a silicon photomultiplier array coupled to an array of high numerical aperture plastic optical fibers to achieve 16-plex detection. The optical fibers ensure efficient light collection while allowing the fixed detector to be mated with diverse sample geometries (e.g., circular or grid), simply by adjusting the fiber configuration. In a head-to-head comparison with a lens-based camera system featuring a cooled detector, the SFCD achieved a 14-fold improved limit of detection in both direct and enzyme-mediated CL reactions. The SFCD also features improved compactness and lower cost, as well as faster temporal resolution compared with camera-based systems while preserving spatial multiplexing and good environmental robustness. Thus, the SFCD has excellent potential for point-of-care biosensing applications

Integration of Multiplexed Microfluidic Electrokinetic Concentrators with a Morpholino Microarray via Reversible Surface Bonding for Enhanced DNA Hybridization

We describe a microfluidic concentration device to accelerate the surface hybridization reaction between DNA and morpholinos (MOs) for enhanced detection. The microfluidic concentrator comprises a single polydimethylsiloxane (PDMS) microchannel onto which an ion-selective layer of conductive polymer poly­(3,4-ethylenedioxythiophene)-poly­(styrenesulfonate) (PEDOT:PSS) was directly printed and then reversibly surface bonded onto a morpholino microarray for hybridization. Using this electrokinetic trapping concentrator, we could achieve a maximum concentration factor of ∼800 for DNA and a limit of detection of 10 nM within 15 min. In terms of the detection speed, it enabled faster hybridization by around 10-fold when compared to conventional diffusion-based hybridization. A significant advantage of our approach is that the fabrication of the microfluidic concentrator is completely decoupled from the microarray; by eliminating the need to deposit an ion-selective layer on the microarray surface prior to device integration, interfacing between both modules, the PDMS chip for electrokinetic concentration and the substrate for DNA sensing are easier and applicable to any microarray platform. Furthermore, this fabrication strategy facilitates a multiplexing of concentrators. We have demonstrated the proof-of-concept for multiplexing by building a device with 5 parallel concentrators connected to a single inlet/outlet and applying it to parallel concentration and hybridization. Such device yielded similar concentration and hybridization efficiency compared to that of a single-channel device without adding any complexity to the fabrication and setup. These results demonstrate that our concentrator concept can be applied to the development of a highly multiplexed concentrator-enhanced microarray detection system for either genetic analysis or other diagnostic assays

Increasing the Detection Sensitivity for DNA-Morpholino Hybridization in Sub-Nanomolar Regime by Enhancing the Surface Ion Conductance of PEDOT:PSS Membrane in a Microchannel

Electrokinetic concentration based on ion concentration polarization (ICP) offers a unique possibility to increase detection sensitivity and speed of surface-based biosensors for low-abundance biomolecules inside a microfluidic channel. To further improve the concentration performance, we investigated the effect of surface ion conductance of the ion-selective conductive polymer membrane, poly­(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS), in a microfluidic channel. By increasing its thickness and surface charge, we could achieve a concentration increase of DNA by <i>6 orders of magnitude</i> from an initial concentration of 100 fM within 10 min. As for the detection via surface hybridization on morpholino (MO) probes, DNA target concentration as low as 10 pM was detected within 15 min. This result means an improvement by <i>2 orders of magnitude</i> in terms of the detection limit compared with our previous developed PEDOT:PSS membrane. These results demonstrate a potential application of the PEDOT:PSS membrane for the ICP-enhanced detection of DNA and other biomolecules in surface-based assays down to picomolar regimes

Inactivation of a putative efflux pump (LmrB) in <i>Streptococcus mutans</i> results in altered biofilm structure and increased exopolysaccharide synthesis: implications for biofilm resistance

<p>Efflux pumps are a mechanism associated with biofilm formation and resistance. There is limited information regarding efflux pumps in <i>Streptococcus mutans</i>, a major pathogen in dental caries. The aim of this study was to investigate potential roles of a putative efflux pump (LmrB) in <i>S. mutans</i> biofilm formation and susceptibility. Upon <i>lmrB</i> inactivation and antimicrobial exposure, the biofilm structure and expression of other efflux pumps were examined using confocal laser scanning microscopy (CLSM) and qRT-PCR. <i>lmrB</i> inactivation resulted in biofilm structural changes, increased EPS formation and EPS-related gene transcription (<i>p</i> < 0.05), but no improvement in susceptibility was observed. The expression of most efflux pump genes increased upon <i>lmrB</i> inactivation when exposed to antimicrobials (<i>p</i> < 0.05), suggesting a feedback mechanism that activated the transcription of other efflux pumps to compensate for the loss of <i>lmrB</i>. These observations imply that sole inactivation of <i>lmrB</i> is not an effective solution to control biofilms.</p

Data_Sheet_1_RNase III coding genes modulate the cross-kingdom biofilm of Streptococcus mutans and Candida albicans.docx

Streptococcus mutans constantly coexists with Candida albicans in plaque biofilms of early childhood caries (ECC). The progression of ECC can be influenced by the interactions between S. mutans and C. albicans through exopolysaccharides (EPS). Our previous studies have shown that rnc, the gene encoding ribonuclease III (RNase III), is implicated in the cariogenicity of S. mutans by regulating EPS metabolism. The DCR1 gene in C. albicans encodes the sole functional RNase III and is capable of producing non-coding RNAs. However, whether rnc or DCR1 can regulate the structure or cariogenic virulence of the cross-kingdom biofilm of S. mutans and C. albicans is not yet well understood. By using gene disruption or overexpression assays, this study aims to investigate the roles of rnc and DCR1 in modulating the biological characteristics of dual-species biofilms of S. mutans and C. albicans and to reveal the molecular mechanism of regulation. The morphology, biomass, EPS content, and lactic acid production of the dual-species biofilm were assessed. Quantitative real-time polymerase chain reaction (qRT-PCR) and transcriptomic profiling were performed to unravel the alteration of C. albicans virulence. We found that both rnc and DCR1 could regulate the biological traits of cross-kingdom biofilms. The rnc gene prominently contributed to the formation of dual-species biofilms by positively modulating the extracellular polysaccharide synthesis, leading to increased biomass, biofilm roughness, and acid production. Changes in the microecological system probably impacted the virulence as well as polysaccharide or pyruvate metabolism pathways of C. albicans, which facilitated the assembly of a cariogenic cross-kingdom biofilm and the generation of an augmented acidic milieu. These results may provide an avenue for exploring new targets for the effective prevention and treatment of ECC.</p