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Rapid and accurate detection of Klebsiella pneumoniae carbapenem resistance is important for infection control and targeted antibiotic therapy. PCR-based assay performance heavily depends on the quality and quantity of template DNA. Challenges arise from the necessity to isolate chromosomal and large plasmid-encoded resistance genes simultaneously from a limited number of target cells and to remove PCR inhibitors. qPCRs for the detection of K. pneumoniae strains carrying blaOXA-48, blaNDM-1, blaKPC-2, and blaVIM-1 carbapenemase genes were developed. We compared the performance of template DNA extracted with silica column-based methods, reversed elution systems, and lysis-only methods either from diluted culture fluid or from a synthetic stool matrix which contained PCR inhibitors typically present in stool. The synthetic stool matrix was chosen to mimic K. pneumoniae containing rectal swabs or stool samples in a reproducible manner. For total DNA isolated from culture fluid, resistance gene detection by qPCR was always possible, independent of the extraction method. However, when total DNA was isolated from synthetic stool matrix spiked with K. pneumoniae, most methods were insufficient. The best performance of template DNA was obtained with reversed elution. This highlights the importance of choosing the right DNA extraction method for consistent carbapenem resistance detection by PCR. [ABSTRACT FROM AUTHOR]

Total internal reflection fluorescence (TIRF) coupled with hydrogel--DNA droplet microarrays covalently bound on PMMA substrates presents a reusable, sensitive platform for evaluating DNA hybridization and for rapid biochip development. Hydrogel microarrays, which contain covalently bound DNA probes, are created via a simple printing and photocross-linking process. TIRF measurements of the arrays display robust reusability, show linear sensitivity down to 5 fmol of fluorescently labeled target DNA, and are sensitive to single basepair mismatches. Additionally, the ability to interrogate larger DNA is shown through studies with PCR amplification hybridization. We conclusively demonstrate an efficient, reproducible, low cost platform for DNA hybridization studies that could be used for fast high-throughput diagnostics as well as biochip development. 10.1021/ac1008578

Nucleic acid amplification techniques such as real-time PCR are essential instruments for the identification and quantification of viruses. They are fast, very sensitive and highly specific, but often require elaborate and labor intensive sample preparation to achieve successful amplification of the target sequence. In this work we demonstrate the complete microfluidic preparation of amplifiable virus DNA from dilute specimens. Our approach combines free-flow electrophoretic preconcentration of viral particles with thermal lysis and gel-electrophoretic nucleic acid extraction on a single device. The on-chip preconcentration achieves a capture efficiency of >99% for dilute suspensions of bacteriophage PhiX174. Following preconcentration, phages are thermally lysed and released DNA is recovered after 40 s of on-chip gel-electrophoresis with a recovery rate of ∼73%. Furthermore we demonstrate a detection limit of ∼1 PFU ml−1 (∼0.02 DNA copies per μl) for the detection of bacteriophage PhiX174 by PCR. To simplify operation of the device, we describe the development of a custom-made chip holder as well as a compact peristaltic pump and power supply, which enable user-friendly operation with low risk of cross-contamination and high potential for automation in the field of point-of-care diagnostics. [ABSTRACT FROM AUTHOR]

The rapid and simultaneous detection of DNA and protein biomarkers is necessary to detect the outbreak of a disease or to monitor a disease. For example, cardiovascular diseases are a major cause of adult mortality worldwide. We have developed a rapidly adaptable platform to assess biomarkers using a microfluidic technology. Our model mimics autoantibodies against three proteins, C-reactive protein (CRP), brain natriuretic peptide (BNP), and low-density lipoprotein (LDL). Cell-free mitochondrial DNA (cfmDNA) and DNA controls are detected via fluorescence probes. The biomarkers are covalently bound on the surface of size- (11–15 μm) and dual-color encoded microbeads and immobilized as planar layer in a microfluidic chip flow cell. Binding events of target molecules were analyzed by fluorescence measurements with a fully automatized fluorescence microscope (end-point and real-time) developed in house. The model system was optimized for buffers and immobilization strategies of the microbeads to enable the simultaneous detection of protein and DNA biomarkers. All prime target molecules (anti-CRP, anti-BNP, anti-LDL, cfmDNA) and the controls were successfully detected both in independent reactions and simultaneously. In addition, the biomarkers could also be detected in spiked human serum in a similar way as in the optimized buffer system. The detection limit specified by the manufacturer is reduced by at least a factor of five for each biomarker as a result of the antibody detection and kinetic experiments indicate that nearly 50 % of the fluorescence intensity is achieved within 7 min. For rapid data inspection, we have developed the open source software digilogger, which can be applied for data evaluation and visualization. [ABSTRACT FROM AUTHOR]

The full potential of the real-time reverse transcription polymerase chain reaction (RT-PCR) as a rapid and accurate diagnostic method is limited by DNA polymerase inhibitors as well as reverse transcriptase inhibitors which are ubiquitous in clinical samples. rTth polymerase has proven to be more resistant to DNA polymerase inhibitors present in clinical samples for DNA detection and also exhibits reverse transcriptase activity in the presence of Mn2+ ions. However, the capacity of rTth polymerase, which acts as DNA polymerase and reverse transcriptase, to detect RNA in the presence of various inhibitors has not been investigated in detail. Herein, the inhibitors originating from various clinical samples such as blood, urine, feces, bodily fluids, tissues and reagents used during nucleic acid extraction were employed to evaluate the capacity of rTth polymerase to detect RNA. The results show that the inhibitors have different inhibitory effects on the real-time RT-PCR reactions by rTth polymerase, and the inhibitory effects are concentration dependent. Additionally, the capacity of rTth polymerase to detect RNA in the presence of various inhibitors is better or at least comparable with its capacity to detect DNA in the presence of various inhibitors. As a consequence, RNA may be directly detected in the presence of co-purified inhibitors or even directly from crude clinical samples by rTth polymerase. [ABSTRACT FROM AUTHOR]

Although soft contact lenses are more widespread, rigid contact lenses have been around for decades and their materials have been greatly improved, especially in terms of oxygen permeability. Protein adsorption and wettability are, however, complex challenges still faced by both soft and rigid contact lenses (CLs). This study aims at improving these two major attributes by means of a low-pressure magnetron enhanced 15 kHz plasma polymerization process. The wide parameter range of the process allowed the tailoring of the surface properties and delivered stable, reproducible plasma coatings. The XPS, FT-IR, WCA, and QCM surface analysis methods were used to study the nanofilms. A contact angle of down to 18° and a protein (lysozyme) adsorption of only 0.2 µg cm−2 were achieved. Some correlations with the more commonly used 'PEO-like' coatings helped to explain the measurement results. [ABSTRACT FROM AUTHOR]

This work deals with the water uptake and swelling behavior of magnetron-enhanced plasma-polymerized nanofilms in an aqueous medium. Those coatings are used in biomedical applications such as implants or contact lenses. The role of water in biomaterial surface science is considered to be of great importance. Water is not only the major molecule in most living organisms (70%) and the carrier of cells but it is also the medium in which biochemical processes take place. Because of its small size and mobility, water is the first molecule to come in contact with a biomaterial in any clinical application. It is believed, that the degree of polymer swelling strongly affects the interaction of proteins with the surface of the polymer, which determines its biocompatibility. Polymers with a high swelling degree show weak enthalpic interactions with proteins, resulting in a protein-repellency. A new approach is applied, which combines three characterization techniques: dynamic contact angle measurements, optical waveguide spectroscopy, and electrochemical impedance spectroscopy. A relation between the composition of the films and the water and salt transport phenomena through the nanofilm was observed. A higher amount of oxygen in the precursor gas ratio in the polymerization process leads to a higher wettability as well as a higher water and salt intrusion. Based on this observation an electrical model was introduced and helped to interpret the experimental results. [ABSTRACT FROM AUTHOR]

We investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drift in dependence on post curing of the materials is characterized. We 3D printed monolithic rtRPA microreactors and subjected the devices to different post curing protocols. Residual autofluorescence and drift, as well as rtRPA kinetics, were then measured in a custom-made mobile temperature-controlled fluorescence reader (mTFR). Furthermore, we investigated the effects of storage on the devices over a 30-day period. Finally, we present the single- and duplex rtRPA detection of both the organism-specific Klebsiella haemolysin (khe) gene and the New Delhi metallo-β-lactamase 1 (blaNDM-1) gene from Klebsiella pneumoniae. Results: No combination of 3D printing resin and post curing protocol completely inhibited the rtRPA reaction. The autofluorescence and fluorescence drift measured were found to be highly dependent on printing material and wavelength. Storage had the effect of decreasing the autofluorescence of the investigated materials. Both khe and blaNDM-1 were successfully detected by single- and duplex-rtRPA inside monolithic rtRPA microreactors printed from NextDent Ortho Clear (NXOC). The reaction kinetics were found to be close to those observed for rtRPA performed in a microcentrifuge tube without the need for mixing during amplification. Singleplex assays for both khe and blaNDM-1 achieved a limit of detection of 2.5 × 101 DNA copies while the duplex assay achieved 2.5 × 101 DNA copies for khe and 2.5 × 102 DNA copies for blaNDM-1. Impact: We expand on the state of the art by demonstrating a technology that can manufacture monolithic microfluidic devices that are readily suitable for rtRPA. The devices exhibit very low autofluorescence and fluorescence drift and are compatible with RPA chemistry without the need for any surface pre-treatment such as blocking with, e.g., BSA or PEG. [ABSTRACT FROM AUTHOR]

We present a simple to operate microfluidic chip system that allows for the extraction of miRNAs from cells with minimal hands-on time. The chip integrates thermoelectric lysis (TEL) of cells with native gel-electrophoretic elution (GEE) of released nucleic acids and uses non-toxic reagents while requiring a sample volume of only 5 μl. These properties as well as the fast process duration of 180 seconds make the system an ideal candidate to be part of fully integrated point-of-care applications for e.g. the diagnosis of cancerous tissue. GEE was characterized in comparison to state-of-the-art silica column (SC) based RNA recovery using the mirVana kit (Ambion) as a reference. A synthetic miRNA (miR16) as well as a synthetic snoRNA (SNORD48) were subjected to both GEE and SC. Subsequent detection by stem-loop RT-qPCR demonstrated a higher yield for miRNA recovery by GEE. SnoRNA recovery performance was found to be equal for GEE and SC, indicating yield dependence on RNA length. Coupled operation of the chip (TEL + GEE) was characterized using serial dilutions of 5 to 500 MCF7 cancer cells in suspension. Samples were split and cells were subjected to either on-chip extraction or SC. Eluted miRNAs were then detected by stem-loop RT-qPCR without any further pre-processing. The extraction yield from cells was found to be up to ~200-fold higher for the chip system under non-denaturing conditions. The ratio of eluted miRNAs is shown to be dependent on the degree of complexation with miRNA associated proteins by comparing miRNAs purified by GEE from heat-shock and proteinase-K based lysis. [ABSTRACT FROM AUTHOR]

We investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drift in dependence on post curing of the materials is characterized. We 3D printed monolithic rtRPA microreactors and subjected the devices to different post curing protocols. Residual autofluorescence and drift, as well as rtRPA kinetics, were then measured in a custom-made mobile temperature-controlled fluorescence reader (mTFR). Furthermore, we investigated the effects of storage on the devices over a 30-day period. Finally, we present the single- and duplex rtRPA detection of both the organism-specific Klebsiella haemolysin ( khe) gene and the New Delhi metallo-β-lactamase 1 ( bla NDM-1 ) gene from Klebsiella pneumoniae . Results: No combination of 3D printing resin and post curing protocol completely inhibited the rtRPA reaction. The autofluorescence and fluorescence drift measured were found to be highly dependent on printing material and wavelength. Storage had the effect of decreasing the autofluorescence of the investigated materials. Both khe and bla NDM-1 were successfully detected by single- and duplex-rtRPA inside monolithic rtRPA microreactors printed from NextDent Ortho Clear (NXOC). The reaction kinetics were found to be close to those observed for rtRPA performed in a microcentrifuge tube without the need for mixing during amplification. Singleplex assays for both khe and bla NDM-1 achieved a limit of detection of 2.5 × 10 1 DNA copies while the duplex assay achieved 2.5 × 10 1 DNA copies for khe and 2.5 × 10 2 DNA copies for bla NDM-1 . Impact: We expand on the state of the art by demonstrating a technology that can manufacture monolithic microfluidic devices that are readily suitable for rtRPA. The devices exhibit very low autofluorescence and fluorescence drift and are compatible with RPA chemistry without the need for any surface pre-treatment such as blocking with, e.g., BSA or PEG.

PCR inhibitors in clinical specimens negatively affect the sensitivity of diagnostic PCR and RT-PCR or may even cause false-negative results. To overcome PCR inhibition, increase the sensitivity of the assays and simplify the detection protocols, simple methods based on quantitative nested real-time PCR and RT-PCR were developed to detect exogenous DNA and RNA directly from large volumes of whole human blood (WHB). Thermus thermophilus (Tth) polymerase is resistant to several common PCR inhibitors and exhibits reverse transcriptase activity in the presence of manganese ions. In combination with optimized concentrations of magnesium ions and manganese ions, Tth polymerase enabled efficient detection of DNA and RNA from large volumes of WHB treated with various anticoagulants. The applicability of these methods was further demonstrated by examining WHB specimens collected from different healthy individuals and those stored under a variety of conditions. The detection limit of these methods was determined by detecting exogenous DNA, RNA, and bacteria spiked in WHB. To the best of our knowledge, direct RNA detection from large volumes of WHB has not been reported. The results of the developed methods can be obtained within 4 hours, making them possible for rapid and accurate detection of disease-causing agents from WHB.

With a view to developing a rapid pathogen detection system utilizing isothermal nucleic acid amplification, the necessary micro-mixing step is innovatively implemented on a chip. Passive laminar flow mixing of two 6.5 μl batches differing in viscosity is performed within a microfluidic chamber. This is achieved with a novel chip space-saving phaseguide design which allows, for the first time, the complete integration of a passive mixing structure into a target chamber. Sequential filling of batches prior to mixing is demonstrated. Simulation predicts a reduction of diffusive mixing time from hours down to one minute. A simple and low-cost fabrication method is used which combines dry film resist technology and direct wafer bonding. Finally, an isothermal nucleic acid detection assay is successfully implemented where fluorescence results are measured directly from the chip after a one minute mixing sequence. In combination with our previous work, this opens up the way towards a fully integrated pathogen detection system in a lab-on-a-chip format.

The lack of sample pre-treatment concepts that are easily automatable, miniaturized and highly efficient for both small volumes and low target concentrations, is one of the key issues that block the road towards effective miniaturized diagnostic instruments. This paper presents a novel, highly efficient and simple method for low-molecular weight RNA extraction using electricity only. Cells are lysed by thermo-electric lysis and RNA is purified using a gel-electrophoretic purification step. The combination of the two steps in one integrated cartridge reduces the time frame between the two steps, thus protecting RNA from enzymatic degradation. A disposable chip solution is proposed using a novel dry film resist laminate technology that allows cheap, large-scale fabrication. The chip contains crucial microfluidic innovations that allow for a simple user interface, reproducible functioning and precise quantification. Phaseguides are invented that allow controlled spatial injection of gel, injection of sample and recovery of extracted RNA. A precise sample volume can be defined by integrating electrophoretic actuation electrodes in the microfluidic chamber. Electrolytic gas bubbles that are the result of constant-current actuation are driven out from the chip by the novel introduction of capillary bubble-expulsion techniques. The extraction approach and the functionality of the chip are demonstrated for Escherichia coli and Streptococcus thermophilus bacteria. Linear extraction behavior is obtained for transfer-messenger RNA down to one colony-forming unit per microlitre, or five colony-forming units per chip. The latter is an increase in extraction efficiency of a factor of 1000 with respect to the commercial extraction kit Ambion Ribopure. The chip shows particularly good performance for extraction of low-molecular weight RNA, thereby eliminating the need for large ribosomal RNA and DNA removal. RNA can be extracted in less than 11 min, being a speed-up of more than a factor of 20 with respect to commercial extraction kits. The presented solution may find broad acceptance and application in drug discovery and clinical diagnostics.