An open microfluidic point-of-care diagnostic (POC) platform will be presented that not only combines the ability to carry out various kinds of immunological, molecular or clinical chemistry tests at the point-of-care, allows for different sample types and can be used with a variety of detection technologies but in particular serves as open platform for users to integrate their own assays. The key advantages are as follows: 1) A standardized microfluidic cartridge architecture, with a common footprint, defined areas for functions allows for a toolbox-like development of a whole family of cartridges all using the same manufacturing tool and validated modules. 2) This finds its correspondence in the instrument which runs the cartridges. Standard footprint and common locations for functional elements makes the instrument versatile and able to run a variety of different assays in one instrument or allows for a rapid adaptation to new assays. 3) From an assay developer standpoint, such platforms with a proven system architecture provide a convenient pathway towards a simplified regulatory approval.
On the presented platform, we have implemented a variety of assays, molecular diagnostics assays for infectious diseases. In case of these assays, the preferred detection method uses fluorescence. For immunoassays, colorimetric measurements with a simpler camera system are possible. In addition, the system also allows for the integration of sensors for detection.
All cartridges include the complete assay flow from sample introduction to read-out. This includes all sample-preparation steps such as sample homogenization, sample lysis and nucleic acid extraction and concentration. In addition, all reagents required are embedded in the cartridge, either in liquid form or as dried reagents in dedicated locations.
Overall, this platform allows especially small and medium-sized diagnostic companies and initiatives a rapid transition from bench to the market at a reasonable cost, significantly less compared to an ab-initio development.
The prevention of toxic pathogens such as mycotoxins is a constant challenge in food production. We present a microfluidics-enabled system for the identification of pathogens in grain dust, thereby allowing for the detection of such pathogen already in the early raw material stage prior further processing. In order to be utilized in such environment, the system has to be compact and rugged. This is realized by miniaturizing both the analytical workflow (competitive fluorescence immunoassay) using a fully integrated microfluidic cartridge as well as the instrument to operate the cartridge. We have been able to show similar performance of microfluidic chip-based assays compared to standard ELISA plates.
KEYWORDS: Lab on a chip, Pathogens, Robotics, Surveillance, Biological and chemical sensing, Control systems, Liquids, Molecular biology, Bacteria, Biological weapons
Remotely-controlled robotic air and ground vehicles (frequently collectively called drones) play a rapidly increasing role not only in military operations but for many civilian tasks ranging from traffic surveillance to disaster monitoring. The possibility to do remote sampling in areas otherwise difficult to reach as well as their low acquisition and operating cost compared to other technical solutions make them attractive platforms for chemical and biological analysis systems. The restrictions on size and weight on these platforms together with the need for a rugged system layout makes lab-on-a-chip technologies the prime candidates to be implemented on such a drone platform. We report on a lab-on-a-chip system for such tasks which consist out of a fully integrated lab-on-a-chip cartridge and the respective control hardware. The cartridge contains all reagents in liquid and dry form required to perform a molecular biology assay to identify bacterial pathogens such as Brucella and Francisella. These zoonotic bacteria are classified as biological agents and cause the (re-)emerging diseases brucellosis and tularemia, which occur also in developed countries in a relevant number of human clinical cases. After proof-of-concept for these pathogens, it is planned to expand the platform to further B-agents.
Current microfluidics-enabled point-of-care diagnostic systems are typically designed specifically for one assay type, e.g. a molecular diagnostic assay for a single disease of a class of diseases. This approach often leads to high development cost and a significant training requirement for users of different instruments. We have developed an open platform diagnostic system which allows to run molecular, immunological and clinical assays on a single instrument platform with a standardized microfluidic cartridge architecture in an automated sample-in answer-out fashion. As examples, a molecular diagnostic assay for tuberculosis, an immunoassay for HIV p24 and a clinical chemistry assay for ALT liver function have been developed and results of their pre-clinical validation are presented.
KEYWORDS: Pathogens, Biological detection systems, System integration, Lab on a chip, Biological research, Molecular biology, Microfluidics, Bacteria, Liquids, Control systems
Lab-on-a-chip systems are very promising approach for a decentralized continuous pathogen monitoring. The overall presented system consisting of a microtiter plate sized consumable and the respective instrument allows for the detection of airborne biological pathogen. The target pathogens to be detected are Yersinia pestis, Francisella tularensis, Burkholderia mallei, Burkholderia pseudomallei, Brucella melitensis, Brucella abortis, Coxiella burnetti, and Bacillus anthracis. Important to stress that the technical platform can be easily expanded to further pathogens.
In this paper the development of a fully integrated system for the 8-plex detection of bacterial pathogens will be presented. Two exceptional features are combined in this device: The overall system allows for the permanent sampling and analysis of airborne biological pathogens and combines the detection of the respective bacteria on molecular and immunological level.
The overall system consisting of chip and instrument and the biological procedures embedded on chip will be presented.
The diverse human HLA (human leukocyte antigen) system is responsible for antigen presentation and recognition. It is essential for the immune system to maintain a stable defense line, but also is also involved in autoimmunity as well as metabolic disease. HLA-haplotype (HLA-B27), for instance, is associated with inflammatory diseases such as Bechterew's disease. The administration of the HIV drug Abacavir in combination with another HLA-haplotype (HLAB57) is associated with severe hypersensitivity reactions. Accordingly, the HLA status has to be monitored for diagnosis or prior to start of therapy. Along this line, a miniaturized microfluidic platform has been developed allowing performing the complete analytical process from “sample-in” to “answer-out” in a point-of-care environment. The main steps of the analytical cascade inside the integrated system are blood cell lysis and DNA isolation, DNA purification, real-time PCR and quantitative monitoring of the rise of a fluorescent signal appearing during the PCR based sequence amplification. All bio-analytical steps were intended to be performed inside one chip and will be actuated, controlled and monitored by a matching device. This report will show that all required processes are established and tested and all device components work well and interact with the functional modules on the chips in a harmonized fashion.
The continuous monitoring of the environment for lethal pathogens is a central task in the field of biothreat detection. Typical scenarios involve air-sampling in locations such as public transport systems or large public events and a subsequent analysis of the samples by a portable instrument. Lab-on-a-chip technologies are one of the promising technological candidates for such a system. We have developed an integrated microfluidic system with automatic sampling for the detection of CBRNE-related pathogens. The chip contains a two-pronged analysis strategy, on the one hand an immunological track using antibodies immobilized on a frit and a subsequent photometric detection, on the other hand a molecular biology approach using continuous-flow PCR with a fluorescence end-point detection. The cartridge contains two-component molded rotary valve to allow active fluid control and switching between channels. The accompanying instrument contains all elements for fluidic and valve actuation, thermal control, as well as the two detection modalities. Reagents are stored in dedicated reagent packs which are connected directly to the cartridge. With this system, we have been able to demonstrate the detection of a variety of pathogen species.
A portable analytical system for the characterization of liquid environmental samples and beverages in food control was realized. The key element is the implementation of contactless conductivity detection on lab-on-a-chip basis ensuring the system to be operated in a label free mode. Typical target molecules such as small ionic species like Li+, Na+, K+, SO4 2- or NO3-, organic acids in wine whose concentration and ratio to each other documents the wine quality, or caffeine or phosphate in coke were detected. Results from sample matrices like various beverages as water, cola, tea, wine and milk, water from heaters, environmental samples and blood will be presented.
KEYWORDS: Microfluidics, Pathogens, Fluorescence spectroscopy, Lab on a chip, Interfaces, Control systems, Liquids, Contamination, Point-of-care devices, Imaging systems
The ability to integrate complete assays on a microfluidic chip helps to greatly simplify instrument requirements and allows the use of lab-on-a-chip technology in the field. A core application for such field-portable systems is the detection of pathogens in a CBRN scenario such as permanent monitoring of airborne pathogens, e.g. in subway stations or hospitals etc. An immunological assay was chosen as method for the pathogen identification. The conceptual approach was its realization as a lab-on-a-chip system, enabling an easy handling of the sample in an automated manner. The immunological detection takes place on an antibody array directly implemented in the microfluidic network. Different immobilization strategies will be presented showing the performance of the system. Central elements of the disposable microfluidic device like fluidic interface, turning valves, liquid introduction and waste storage, as well as the architecture of measurement and control fluidic network, will be introduced. Overall process times of about 30 minutes were achieved and assays for the detection of Francisella tularensis and Yersinia pestis are presented. An important feature of the integrated lab-on-a-chip approach is that all waste liquids remain on-chip and contamination risks can be avoided.
In case of transplantation or the identification of special metabolic diseases like coeliac disease, HLA typing has to be done
fast and reliably with easy-to-handle devices by using limited amount of sample. Against this background a lab-on-a-chip
device was realized enabling a fast HLA typing via miniaturized Real-time PCR. Hereby, two main process steps were
combined, namely the extraction of DNA from whole blood and the amplification of the target DNA by Real-time PCR
giving rise-to a semi-quantitative analysis. For the implementation of both processes on chip, a sample preparation and a
real-time module were used. Sample preparation was carried out by using magnetic beads that were stored directly on chip
as dry powder, together with all lysis reagents. After purification of the DNA by applying a special buffer regime, the
sample DNA was transferred into the PCR module for amplification and detection. Coping with a massively increased
surface-to-volume ratio, which results in a higher amount of unspecific binding on the chip surface, special additives
needed to be integrated to compensate for this effect. Finally the overall procedure showed a sensitivity comparable to
standard Real-time PCR but reduced the duration of analysis to significantly less than one hour. The presented work
demonstrates that the combination of lab-on-a-chip PCR with direct optical read-out in a real-time fashion is an extremely
promising tool for molecular diagnostics.
A central method in a standard biochemical laboratory is represented by the polymerase chain reaction (PCR), therefore
many attempts have been performed so far to implement this technique in lab-on-a-chip (LOC) devices. PCR is an ideal
candidate for miniaturization because of a reduction of assay time and decreased costs for expensive bio-chemicals. In
case of the “classical” PCR, detection is done by identification of DNA fragments electrophoretically separated in
agarose gels. This method is meanwhile frequently replaced by the so-called Real-Time-PCR because here the
exponential increase of amplificates can be observed directly by measurement of DNA interacting fluorescent dyes. Two
main methods for on-chip PCRs are available: traditional “batch” PCR in chambers on a chip using thermal cycling,
requiring about 30 minutes for a typical PCR protocol and continuous-flow PCR, where the liquid is guided over
stationary temperature zones. In the latter case, the PCR protocol can be as fast as 5 minutes. In the presented work, a
proof of concept is demonstrated for a real-time-detection of PCR products in microfluidic systems.
The ability to integrate complete assays on a microfluidic chip helps to greatly simplify instrument requirements and allows the use of lab-on-a-chip technology in the field. A core application for such field-portable systems is the detection of pathogens in a CBRNE scenario such as permanent monitoring of airborne pathogens, e.g. in metro stations or hospitals etc. As one assay methodology for the pathogen identification, enzymatic assays were chosen. In order evaluate different detection strategies, the realized on-chip enzyme assay module has been designed as a general platform chip. In all application cases, the assays are based on immobilized probes located in microfluidic channels. Therefore a microfluidic chip was realized containing a set of three individually addressable channels, not only for detection of the sample itself also to have a set of references for a quantitative analysis. It furthermore includes two turning valves and a waste container for clear and sealed storage of potential pathogenic liquids to avoid contamination of the environment. All liquids remain in the chip and can be disposed of in proper way subsequently to the analysis. The chip design includes four inlet ports consisting of one sample port (Luer interface) and three mini Luer interfaces for fluidic support of e.g. washing buffer, substrate and enzyme solution. The sample can be applied via a special, sealable sampling vessel with integrated female Luer interface. Thereby also pre-anaytical contamination of the environment can be provided. Other reagents that are required for analysis will be stored off chip.
Aim of the paper is the orientation of research and development on a completely new approach to innovative in-field and point of care diagnostics in industry, biology and medicine. Central functional modules are smartphones and/or smart pads supplemented by additional hardware apps and software apps. Specific examples are given for numerous practical applications concerning optodigital instrumentations. The methodical classification distinguishes between different levels for combination of hardware apps (hwapps) and software apps (swapps) with smartphones and/or smartpads. These methods are fundamental enablers for the transformation from stationary conventional laboratory diagnostics into mobile innovative in-field and point of care diagnostics. The innovative approach opens so far untapped enormous markets due to the convenience, reliability and affordability of smartphone and/or smartpad instruments. A highly visible advantage of smartphones and/or smartpads is the huge number of their distribution, their worldwide connectivity via cloud services and the experienced capability of their users for practical operations.
The translation of bio-analytical processes into an automatically functioning microfluidic platform is an attractive task
for several reasons. However, due to the complexity of the resulting integrated device covering various process steps like
lysis, DNA extraction and purification, continuous-flow PCR and detection etc., these single functional units have to be
carefully developed and evaluated in a first step, thus allowing a functional verification prior to final device integration.
All the modules as well as the final integrated device have to be manufactured using scalable industrial manufacturing
methods, namely injection molding, in order to facilitate commercialization The final integrated device should have a
footprint such as SBS-titerplate format, which is generally excepted by the user. For optimal space utilization,
microfluidic structures should be on both the top and the bottom side of the device connected with through-holes. The
device described in this report is a pathogen DNA analysis example realising all the above prerequisites. Sample is
introduced through a Luer-connector in one corner. DNA is extracted in a chamber, which is filled with magnetic beads.
All necessary liquid reagents are stored in 500μl modules which are clipped onto the chip. The sample is aliquoted into 8
tracks, liquefies the PCR-reagents which are stored in lyophilized form in small chambers and runs through a
meandering channel, in which continuous-flow PCR takes place. The samples are then transferred to the top of the chip
and run back to the detection zone where a fluorescence detection of the PCR-products takes place before flowing into
waste. As in the device an 8-plex detection is targeted, the spacing of the microchannels after qPCR had to be very
narrow in order to have all channels within the field of vision of the detection system.
Microfluidics has proven to be a very effective technology for the identification of biological and chemical analytes in a
CBRNE scenario. As it will be shown in the following, the required steps of those analytical processes are manifold
making the development of an integrated microfluidic device a complicated project with a high level of technological
risk, because all necessary analytical processes have to be implemented into a single device. The implementation is
initiated by a dissection of the biochemical workflow into mandatory bio-analytical steps and the resulting protocol for
each of those steps is translated into an appropriate design of a chip-based unit. In this report, examples for such chipbased
functional modules are given. In addition, examples for a merging of positively tested modules into an integrated
chip are shown and, finally, representatives for a smooth interaction between outer world, microfluidic chip, and chip
driving instrument are presented.
There is a need to design an integrated microfluidic platform as simple and lean as possible in order to meet the
requirements for a miniaturized system. Magnetic particles show a great versatility in performing several of the functions
necessary in many microfluidic assays. We therefore have developed a compact portable system to perform magneticbead-
based sample preparation steps in a chip such as DNA-extraction or particle-enhanced mixing of reagents. A
central application in a standard biochemical/biological/medical laboratory is represented by PCR. The execution of a
cyclic heating profile during PCR is a considerable stress for chip and liquid inside the chip because evaporation and
uncontrolled condensation or unintended motion of the PCR solution.
One strategy to overcome this problem consists of the implementation of valves flanking a stationary PCR in appropriate
incubation cavities. In addition to the well-known elastomeric membrane valves, wax-valves mechanical turning or
rotary valves flanking the PCR chamber, we present in this paper the use of clustered magnetic particles as blocking
valves for such reaction chambers.
We report on the capability of assembled magnetic particles to act as rather simple configurated valves during a PCR
typical temperature regime. These novel valves efficiently withstand 1.5 bar pressure, prevent loss of aqueous liquid
inside the reaction chamber via evaporation or bubble formation, and do not express adverse effects on any biological
reaction inside the chip-based PCR cavity. The latter properties have been proven by a set of different PCRs performed
in chip-based cavities.
For complex biological or diagnostic assays, the development of an integrated microfluidic device can be difficult and
error-prone. For this reason, a modular approach, using individual microfluidic functional modules for the different
process steps, can be advantageous. However often the interconnection of the modules proves to be tedious and the
peripheral instrumentation to drive the various modules is cumbersome and of large size. For this reason, we have
developed an integrated instrument platform which has generic functionalities such as valves and pumps, heating zones
for continuous-flow PCR, moveable magnets for bead-based assays and an optical detection unit build into the
instrument. The instrument holds a titerplate-sized carrier in which up to four microscopy-slide sized microfluidic
modules can be clipped in. This allows for developing and optimizing individual assay steps without the need to modify
the instrument or generate a completely new microfluidic cartridge.
As a proof-of-concept, the automated sample processing of liquor or blood culture in microfluidic structures for
detection of currently occuring Neisseria meningitidis strains was carried out. This assay involves the extraction of
bacterial DNA, the fluorescent labeling, amplification using PCR as well as the hybridization of the DNA molecules in
three-dimensional capture sites spotted into a microchannel. To define the assay sensitivity, chip modules were tested
with bacteria spiked samples of different origins and results were controlled by conventional techniques. For liquor or
blood culture, the presence of 200 bacteria was detected within 1 hour.
In this paper we demonstrate the development of an integrated lab-on-a-chip system for the point-of-care diagnostics of
Coeliac disease. A two-step approach is used, using two different microfluidic chips with identical footprint and
functional landscape, one for the analysis of the genetic predisposition using human leukocyte antigen typing, the second
for a serology assay. Emphasis has been put on using a seamless technology path from prototyping to final device
manufacture in order to allow an upscaling of production volumes without a chance in production technology. Therefore,
injection molding has been extensively used, however using standard formats allowing the use of family tools in order to
reduce the cost of manufacturing.
Taking advantage of microfluidics technology, a Lab-on-Chip system was developed offering the possibility of
performing HLA (Human Leukocyte Antigen) typing to test genetic predisposition to coeliac disease and measure the
level of immunodeficiency at the point-of-care. These analysis procedures are implemented on two different microfluidic
cartridges, both having identical interfacial connections to the identical automated instrument.
In order to assess the concentration of the targeted analytes in human blood, finger prick samples are processed to either
extract genomic DNA carrying the coeliac disease gene or blood plasma containing the disease specific antibodies. We
present here the different microfluidic modules integrated in a common platform, capable of automated sample
preparation and analyte detection.
In summary, this new microfluidic approach will dramatically reduce the costs of materials (polymer for the disposable
chips and minute amount of bio-reagents) and minimize the time for analysis down to less than 20 minutes. In
comparison to the state of the art detection of coeliac disease this work represents a tremendous improvement for the
patient's quality of live and will significantly reduce the cost burden on the health care system.
Lab-on-a-chip systems are seen as a very promising approach for a decentralized continuous pathogen monitoring
technology. In this paper, we present the development of a fully integrated device for the multiplexed nucleic-acid based
identification of pathogens. Due to the complexity of such a fully integrated device, in a first development step,
functional modules for the various process steps like lysis, DNA extraction and purification, continuous-flow PCR and
detection have been developed and evaluated, allowing a functional verification prior to integration. All the modules as
well as the final integrated device have been manufactured using scaleable industrial manufacturing methods, namely
injection molding in order to facilitate commercialization.
The overall aim is the realization of a reliable, ultrafast, and portable tool for the identification of B-agents at the point
of interest. PCR is the method to be used for the doubtless identification of e.g. bacteria, and viruses. Miniaturization is
the way to include the overall analysis process, from sample preparation to detection, on a microtiterplate-sized
consumable device and to allow to carry out the analysis without the need for an equipped biological laboratory. The
novel PCR concept with constant temperature zones allows also for the instrument to become portable, due to much
reduced power consumption since no thermocycling is necessary. This paper focuses on the overall concept to
implement the biological reactions for the analysis on-chip, the methods of sample preparation on-chip, and the results
of the ultrafast PCR with B-agents on-chip, as well as the basic instrument.
We present a compact portable chip-based capillary electrophoresis system that employs capacitively coupled contactless
conductivity detection (C4D) operating at 4 MHz as an alternative detection method compared to the commonly used
optical detection based on laser-induced fluorescence. Emphasis was put on system integration and industrial
manufacturing technologies for the system. Therefore, the disposable chip for this system is fabricated out of PMMA
using injection molding; the electrodes are screen-printed or thin-film electrodes. The system is designed for the
measurement of small ionic species like Li+, Na+, K+, SO42- or NO3- typically present in foods like milk and mineral
water as well as acids e.g. in wine.
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.
Continuous-flow PCR has proven to be a powerful method for the amplification of genetic material due to its high speed
and the possibility to perform amplicon detection and separation on-chip. A unique possibility of this method is the
simultaneous amplification of several samples within a single chip by sample stacking, either having identical samples in
several sample plugs separated by e.g. a mineral oil or using different samples in each sample plug. We have
demonstrated the viability of sample stacking with a commercially available continuous-flow PCR system with a variety
of protocols and samples. Further integration steps like thermal lysis and on-chip lyophilisate storage have been
performed, with subsequent successful PCR. Chip modules for DNA extraction either with magnetic beads or membrane
filters have been developed.
We present a compact portable chip-based capillary electrophoresis system that employs capacitively coupled contactless
conductivity detection (C4D) operating at 4 MHz as an alternative detection method compared to the commonly used
optical detection employing laser-induced fluorescence. The disposable chip for this system is fabricated out of PMMA
using injection molding; the electrodes are screen-printed or thin-film electrodes. The system allows the measurement of
small ions like Li, Na, K typically present in foodstuff like milk and mineral water as well as acids in wine.
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