CENTRIFUGAL LABTUBE FOR FULLY AUTOMATED DNA EXTRACTION & LAMP AMPLIFICATION BASED ON AN INTEGRATED, LOW-COST HEATING SYSTEM

In this paper, we introduce a disposable battery-driven heating system for loop-mediated isothermal DNA amplification (LAMP) inside a centrifugally-driven DNA-extraction platform (LabTube). We demonstrate fully automated, fully closed extraction of as little as 100 DNA copies of verotoxin-producing (VTEC) Escherichia coli lysate in water, milk and apple juice in a standard laboratory centrifuge, followed by subsequent automatic LAMP amplification with an overall time-to-result of 1.5hrs. The system is disposable, fully closed and automated, requiring only a single pipetting step. The microcontroller-driven heating system is low-cost (<1$) and it can be easily parallelized. Because the heated LabSystem runs within a standard laboratory centrifuge, it is suitable for DNA extraction and amplification in low-resource areas, at production sites or sales locations

Centrifugal LabTube platform for fully automated DNA purification and LAMP amplification based on an integrated, low-cost heating system

This paper introduces a disposable battery-driven heating system for loop-mediated isothermal DNA amplification (LAMP) inside a centrifugally-driven DNA purification platform (LabTube). We demonstrate LabTube-based fully automated DNA purification of as low as 100 cell-equivalents of verotoxin-producing Escherichia coli (VTEC) in water, milk and apple juice in a laboratory centrifuge, followed by integrated and automated LAMP amplification with a reduction of hands-on time from 45 to 1 min. The heating system consists of two parallel SMD thick film resistors and a NTC as heating and temperature sensing elements. They are driven by a 3 V battery and controlled by a microcontroller. The LAMP reagents are stored in the elution chamber and the amplification starts immediately after the eluate is purged into the chamber. The LabTube, including a microcontroller-based heating system, demonstrates contamination-free and automated sample-to-answer nucleic acid testing within a laboratory centrifuge. The heating system can be easily parallelized within one LabTube and it is deployable for a variety of heating and electrical applications

Capillary-Driven Pumping for Passive Degassing and Fuel Supply in Direct Methanol Fuel Cells

Abstract In this paper we present a new concept of creating and using capillary pressure gradients for passive degassing and passive methanol supply in direct methanol fuel cells (DMFCs). An anode flow field consisting of parallel tapered channels structures is applied to achieve the passive supply mechanism. The flow is propelled by the surface forces of deformed CO 2 bubbles, generated as a reaction product during DMFC operation. This work focuses on studying the influence of channel geometry and surface properties on the capillary-induced liquid flow rates at various bubbly gas flow rates. Besides the aspect ratios and opening angles of the tapered channels, the static contact angle as well as the effect of contact angle hysteresis has been identified to significantly influence the liquid flow rates induced by capillary forces at the bubble menisci. Applying the novel concept, we show that the liquid flow rates are up to thirteen times higher than the methanol oxidation reaction on the anode requires. Experimental results are presented that demonstrate the continuous passive operation of a DMFC for more than 15 h

Fully automated point-of-care differential diagnosis of acute febrile illness

Background In this work, a platform was developed and tested to allow to detect a variety of candidate viral, bacterial and parasitic pathogens, for acute fever of unknown origin. The platform is based on a centrifugal microfluidic cartridge, the LabDisk (“FeverDisk” for the specific application), which integrates all necessary reagents for sample-to-answer analysis and is processed by a compact, point-of-care compatible device. Methodology/Principal findings A sample volume of 200 μL per FeverDisk was used. In situ extraction with pre-stored reagents was achieved by bind-wash-elute chemistry and magnetic particles. Enzymes for the loop-mediated isothermal amplification (LAMP) were pre-stored in lyopellet form providing stability and independence from the cold chain. The total time to result from sample inlet to read out was 2 h. The proof-of-principle was demonstrated in three small-scale feasibility studies: in Dakar, Senegal and Khartoum, Sudan we tested biobanked samples using 29 and 9 disks, respectively; in Reinfeld, Germany we tested spiked samples and analyzed the limit of detection using three bacteria simultaneously spiked in whole blood using 15 disks. Overall during the three studies, the FeverDisk detected dengue virus (different serotypes), chikungunya virus, Plasmodium falciparum, Salmonella enterica Typhi, Salmonella enterica Paratyphi A and Streptococcus pneumoniae. Conclusions/Significance The FeverDisk proved to be universally applicable as it successfully detected all different types of pathogens as single or co-infections, while it also managed to define the serotype of un-serotyped dengue samples. Thirty-eight FeverDisks at the two African sites provided 59 assay results, out of which 51 (86.4%) were confirmed with reference assay results. The results provide a promising outlook for future implementation of the platform in larger prospective clinical studies for defining its clinical sensitivity and specificity. The technology aims to provide multi-target diagnosis of the origins of fever, which will help fight lethal diseases and the incessant rise of antimicrobial resistance.Additional co-authors: Sieghard Frischmann, Konstantinos Mitsakaki

Centrifugal Step Emulsification can Produce Water in Oil Emulsions with Extremely High Internal Volume Fractions

The high throughput preparation of emulsions with high internal volume fractions is important for many different applications, e.g., drug delivery. However, most emulsification techniques reach only low internal volume fractions and need stable flow rates that are often difficult to control. Here, we present a centrifugal high throughput step emulsification disk for the fast and easy production of emulsions with high internal volume fractions above 95%. The disk produces droplets at generation rates of up to 3700 droplets/s and, for the first time, enables the generation of emulsions with internal volume fractions of &gt;97%. The coefficient of variation between droplet sizes is very good (4%). We apply our system to show the in situ generation of gel emulsion. In the future, the recently introduced unit operation of centrifugal step emulsification may be used for the high throughput production of droplets as reaction compartments for clinical diagnostics or as starting material for micromaterial synthesis

Magnetophoresis in Centrifugal Microfluidics at Continuous Rotation for Nucleic Acid Extraction

Centrifugal microfluidics enables fully automated molecular diagnostics at the point-of-need. However, the integration of solid-phase nucleic acid extraction remains a challenge. Under this scope, we developed the magnetophoresis under continuous rotation for magnetic bead-based nucleic acid extraction. Four stationary permanent magnets are arranged above a cartridge, creating a magnetic field that enables the beads to be transported between the chambers of the extraction module under continuous rotation. The centrifugal force is maintained to avoid uncontrolled spreading of liquids. We concluded that below a frequency of 5 Hz, magnetic beads move radially inwards. In support of magnetophoresis, bead inertia and passive geometrical design features allow to control the azimuthal bead movement between chambers. We then demonstrated ferrimagnetic bead transfer in liquids with broad range of surface tension and density values. Furthermore, we extracted nucleic acids from lysed Anopheles gambiae mosquitoes reaching comparable results of eluate purity (LabDisk: A260/A280 = 1.6 &plusmn; 0.04; Reference: 1.8 &plusmn; 0.17), and RT-PCR of extracted RNA (LabDisk: Ct = 17.9 &plusmn; 1.6; Reference: Ct = 19.3 &plusmn; 1.7). Conclusively, magnetophoresis at continuous rotation enables easy cartridge integration and nucleic acid extraction at the point-of-need with high yield and purity

Centrifugal Microfluidic Integration of 4-Plex ddPCR Demonstrated by the Quantification of Cancer-Associated Point Mutations

We present the centrifugal microfluidic implementation of a four-plex digital droplet polymerase chain reaction (ddPCR). The platform features 12 identical ddPCR units on a LabDisk cartridge, each capable of generating droplets with a diameter of 82.7 &plusmn; 9 &micro;m. By investigating different oil&ndash;surfactant concentrations, we identified a robust process for droplet generation and stabilization. We observed high droplet stability during thermocycling and endpoint fluorescence imaging, as is required for ddPCRs. Furthermore, we introduce an automated process for four-color fluorescence imaging using a commercial cell analysis microscope, including a customized software pipeline for ddPCR image evaluation. The applicability of ddPCRs is demonstrated by the quantification of three cancer-associated KRAS point mutations (G12D, G12V and G12A) in a diagnostically relevant wild type DNA background. The four-plex assay showed high sensitivity (3.5&ndash;35 mutant DNA copies in 15,000 wild type DNA copies) and linear performance (R&sup2; = 0.99) across all targets in the LabDisk

A versatile-deployable bacterial detection system for food and environmental safety based on LabTube-automated DNA purification, LabReader-integrated amplification, readout and analysis

Contamination of foods is a public health hazard that episodically causes thousands of deaths and sickens millions worldwide. To ensure food safety and quality, rapid, low-cost and easy-to-use detection methods are desirable. Here, the LabSystem is introduced for integrated, automated DNA purification, amplification and detection. It consists of a disposable, centrifugally driven DNA purification platform (LabTube) and a low-cost UV/vis-reader (LabReader). For demonstration of the LabSystem in the context of food safety, purification of Escherichia coli (non-pathogenic E. coli and pathogenic verotoxin-producing E. coli (VTEC)) in water and milk and the product-spoiler Alicyclobacillus acidoterrestris (A. acidoterrestris) in apple juice was integrated and optimized in the LabTube. Inside the LabReader, the purified DNA was amplified, readout and analyzed using both qualitative isothermal loop-mediated DNA amplification (LAMP) and quantitative real-time PCR. For the LAMP-LabSystem, the combined detection limits for purification and amplification of externally lysed VTEC and A. acidoterrestris are 10[superscript 2]–10[superscript 3] cell-equivalents. In the PCR-LabSystem for E. coli cells, the quantification limit is 10[superscript 2] cell-equivalents including LabTube-integrated lysis. The demonstrated LabSystem only requires a laboratory centrifuge (to operate the disposable, fully closed LabTube) and a low-cost LabReader for DNA amplification, readout and analysis. Compared with commercial DNA amplification devices, the LabReader improves sensitivity and specificity by the simultaneous readout of four wavelengths and the continuous readout during temperature cycling. The use of a detachable eluate tube as an interface affords semi-automation of the LabSystem, which does not require specialized training. It reduces the hands-on time from about 50 to 3 min with only two handling steps: sample input and transfer of the detachable detection tube

Automated Pre-Analytic Processing of Whole Saliva Using Magnet-Beating for Point-of-Care Protein Biomarker Analysis

Saliva offers many advantages for point-of-care (PoC) diagnostic applications due to non-invasive, easy, and cost-effective methods of collection. However, the complex matrix with its non-Newtonian behavior and high viscosity poses handling challenges. Several tedious and long pre-analytic steps, incompatible with PoC use, are required to liquefy and homogenize saliva samples before protein analysis can be performed. We apply magnet-beating to reduce hands-on time and to simplify sample preparation. A magnet in a chamber containing the whole saliva is actuated inside a centrifugal microfluidic cartridge by the interplay of centrifugal and magnetic forces. Rigorous mixing, which homogenizes the saliva sample, is then initiated. Consequently, fewer manual steps are required to introduce the whole saliva into the cartridge. After 4 min of magnet-beating, the processed sample can be used for protein analysis. The viscosity of whole saliva has been reduced from 10.4 to 2.3 mPa s. Immunoassay results after magnet-beating for three salivary periodontal markers (MMP-8, MMP-9, TIMP-1) showed a linear correlation with a slope of 0.99 when compared to results of reference method treated samples. Conclusively, magnet-beating has been shown to be a suitable method for the pre-analytic processing of whole saliva for fully automated PoC protein analysis