Rainer Gransee

Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n = 28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.

Automated microfluidically controlled electrochemical biosensor for the rapid and highly sensitive detection of Francisella tularensis.

Tularemia is a highly infectious zoonotic disease caused by a Gram-negative coccoid rod bacterium, Francisella tularensis. Tularemia is considered as a life-threatening potential biological warfare agent due to its high virulence, transmission, mortality and simplicity of cultivation. In the work reported here, different electrochemical immunosensor formats for the detection of whole F. tularensis bacteria were developed and their performance compared. An anti-Francisella antibody (FB11) was used for the detection that recognises the lipopolysaccharide found in the outer membrane of the bacteria. In the first approach, gold-supported self-assembled monolayers of a carboxyl terminated bipodal alkanethiol were used to covalently cross-link with the FB11 antibody. In an alternative second approach F(ab) fragments of the FB11 antibody were generated and directly chemisorbed onto the gold electrode surface. The second approach resulted in an increased capture efficiency and higher sensitivity. Detection limits of 4.5 ng/mL for the lipopolysaccharide antigen and 31 bacteria/mL for the F. tularensis bacteria were achieved. Having demonstrated the functionality of the immunosensor, an electrode array was functionalised with the antibody fragment and integrated with microfluidics and housed in a tester set-up that facilitated complete automation of the assay. The only end-user intervention is sample addition, requiring less than one-minute hands-on time. The use of the automated microfluidic set-up not only required much lower reagent volumes but also the required incubation time was considerably reduced and a notable increase of 3-fold in assay sensitivity was achieved with a total assay time from sample addition to read-out of less than 20 min.

SmartHEALTH: a microfluidic multisensor platform for POC cancer diagnostics

A universal microfluidic platform as a multisensor device for cancer diagnostics, developed within the framework of the EU project SmartHEALTH [1], will be presented. Based on a standardization concept, a microfluidic platform was realized that contains various functional modules in order to allow in its final setup to run a complete diagnostic assay on a chip starting with sample preparation to a final detection via a sensor array. A twofold concept was pursued for the development and standardization: On the one hand, a standard footprint with defined areas for special functional elements was chosen, on the other hand a toolbox-approach [2] was used whereas in a first instance different functional fluidic modules were realized, evaluated and afterwards integrated into the microfluidic multisensor platform. One main characteristic of the platform is that different kind of sensors can be used with the same fluidic chip. For the read-out and fluidic control of the chip, common fluidic interfaces to the instrument were defined. This microfluidic consumable is a hybrid system consisting of a polymer component with an integrated sensor, reagent storage on chip, integrated valves and metering elements.

Fluorescence detection in Lab-on-a-chip systems using ultrafast nucleic acid amplification methods

Today, nucleic amplification plays a key role in modern molecular biology allowing fast and specific laboratory diagnostics testing. An ultrafast microfluidic module (allowing 30 polymeric chain reaction (PCR) cycles in 6 minutes) based on an oscillating fluid plug concept was previously developed[1]. This system allows the amplification of native genomic deoxyribonucleic acid molecules (DNA) even from whole blood samples but still lacks some functionality compared to commercial bench top systems. This work presents the actual status of the renewed and advanced system, permitting the automated optical detection of not only the fluid plug position but also fluorescence detection. The system uses light emitting diodes (LED) for illumination and a low cost CMOS web-camera for optical detection. Image data processing allows the automated process control of the overall system components. Therefore, the system enables the performance of rapid and robust nucleic acid amplifications together with the integration of real time measurement technology. This allows the amplification and simultaneous quantification of the DNA molecules. The possibility to integrate swift nucleic amplification and optical detection into complex sample-to-answer analysis platforms opens up new pathways towards fast and transportable low-cost point of care devices.

Effect of surface structuring onto the efficiency of the in- and out-coupling of light from a chip in Lab-on-a-chip approaches with optical detection

Optical detection methods have been implemented on micro-fluidic chips containing channels or cavities of different geometries e.g. for colorimetry or fluorescence measurements with excitation within the chip plane [1-2]. The most prominent problem of the read-out from a micro-fluidic chip is the limitation of the optical yield. Without e.g. an immersion liquid for compensation of the total reflection on the boundary, only about 11-13% of rays cross over the boundary from a polymer chip to air. One efficient method to increase the optical yield from a chip is a ray reorientation inside of the chip using an additional surface structure creating new incident refraction conditions on the boundary before rays are leaving the chip. The use of 45°-tilted mirror arrangements for in- and out-coupling of the fluorescence signal from a micro-fluidic chip and the realization of this principle for low-cost fluorescence detection systems have been published [3]. This paper includes the investigation of the effect of different tilt angles of total reflection and metallized-surface mirrors for an analyte volume emitter, using the ray-tracing simulation tool OptiCAD10. Furthermore, an estimation of the influence of a surface-emitted signal for different geometries of metallized detection cells with or without a combination with external lenses on the out-coupling efficiency will be presented. The best result of an out-coupling efficiency increase of 10 times was achieved for a combination of a structured and metallized detection cell with an external cylindrical lens.

Active integrated components for fluid control in automatic analytical chip-based systems

A robust optical sensor for liquid control in fluidic channels is reported. The sensor operates on light intensity modulation resulting from alteration of total internal reflection into partial reflection. When a liquid guided in a channel covers an integrated prism, the total internal reflection is changed into a partial reflection, resulting in an intensity modulation of the reflected light. The set-up comprises a fibre which is built in a coupler unit with integrated LED and photodiode as well as a prism micro-machined directly into a micro-fluidic polymeric channel by laser ablation. The Prism is of 45-90-45° type with a dimension of 0.5 mm × 1 mm × 2 mm. In this design the radiation of the LED light source is transmitted and collected from the prism by a 50:50 fibre coupler by means of total or partial internal reflection. The sensor was characterised by filling alternately the channel with water and air. The influence of stray light onto the sensor signal was tested by applying a strong uncollimated illumination of the channel. Only a small increase in the output signal level in the presence of air but a strong increase in case of the presence of water could be detected.

Ultrafast real-time PCR with integrated melting curve analysis and duplex capacities using a low-cost polymer lab-on-a-chip system

Nucleic amplification using quantitative polymeric chain reaction (qPCR) has become the gold standard of molecular testing. These systems offer both amplification and simultaneous fluorescence detection. An ultrafast microfluidic module (allowing 30 PCR cycles in 6 minutes) based on the oscillating fluid plug concept was previously developed [1,2] allowing the amplification of native genomic deoxyribonucleic acid (DNA) molecules. This abstract presents the actual status of the advanced system. The upgraded system generates high quality qPCR amplification plots and additional sensitive melting point analysis comparable to data obtained from commercial real-time cyclers. These features provide the user with all information needed to analyze PCR products. The system uses light emitting diodes (LED) for illumination and a low cost Charge-coupled Device (CCD) camera for optical detection. Image data processing allows the automated process control of the overall system components. The system enables the performance of rapid and robust nucleic acid amplifications together with the integration of real time measurement technology. This allows the amplification and simultaneous quantification of the targeted pathogens. The integration of duplex amplification performance allows the incorporation of the necessary controls into the device to validate the PCR performance. This demonstrator can be run either as fully autonomously working device or as OEM part of a sample-to-answer platform.

Automated DNA-preparation system for bacteria out of air sampler liquids

Preventing bacterial contaminations is a significant challenge in applications across a variety of industries, e.g. in food processing, the life sciences or biohazard detection. Here we present a fully automated lab-on-a-chip system wherein a disposable microfluidic chip moulded by polymeric injection is inserted into an operating device. Liquid samples, here obtained from an air sampler, can be processed to extract and lyse bacteria, and subsequently to purify their DNA using a silica matrix. After the washing and elution steps, the DNA solution is dispensed into a reaction vessel for further analysis in a conventional laboratory polymerase chain reaction (PCR) device. We demonstrate the workability and efficiency of our approach with results from a 9 ml liquid sample spiked with E. coli.