SPIE Proceedings [SPIE SPIE Defense, Security, and Sensing - Baltimore, Maryland (Monday 23 April...

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

Rainer Gransee1, Tina Röser1, Klaus Stefan Drese1, Dominik Düchs1, Claudia Disqué2, Gudrun

Zoll3, Stefan Köhne3, and Marion Ritzi-Lehnert1

1 Institut für Mikrotechnik Mainz GmbH, Carl-Zeiss-Straße 18-20, 55129 Mainz, Germany 2 Molzym GmbH Co.KG, Mary-Astell-Strasse 10, 28359 Bremen Germany 3 Wehrwissenschaftliches Institut für Schutztechnologien, Humboldtstraße, 29633 Munster, Germany ABSTRACT 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. 1. INTRODUCTION The detection of biological contamination is instrumental to many applications in food control, the life sciences[1,2], or, increasingly, the quest for effective counterterrorism measures. Conventional laboratory methods require a high degree of costly and time-consuming manual operation by highly trained personnel. Cumbersome modes of transporting samples from their point of origination (e.g. point of care in biomedical applications) to testing centres further impede and delay the analytic process.

Lab-on-a-chip devices ideally integrate all steps necessary for automatic sample analysis. By eliminating the need for human surveillance, they open up a pathway to providing faster, cheaper, safer and more reliable detection of hazardous biological substances[3,4]. Further, portable devices may aid in reducing total processing times by facilitating the physical transport of samples[5,6].

We here present a disposable lab-on-a-chip (LOC) system capable of extracting bacterial DNA from a 9 ml liquid sample. This liquid could, for example, be obtained directly from an air sampler, therefore this sample preparation method can be used to detect biological agents not only in liquid samples but (indirectly) in gaseous ones, as well. Automatic system control is achieved using an external electronic control unit coupled to an on-board microelectronic processor whose purpose is to steer an electromechanical platform. In a first step, this set-up allows for the complete and automatic preparation of an aqueous sample from its injection into the chip to the elution of 130µl of purified DNA into a reaction vessel. The DNA can then be inserted into conventional laboratory instruments like (real-time) polymerase chain reaction (PCR) thermocyclers for further analysis. The whole sample preparation procedure is currently on par with standard laboratory methods in terms of the time needed for sample preparation.

In the following, we first describe manufacturing and technical aspects of our sample preparation system. Then we present results showing the experimental validation of the system using different concentration of bacteria and give an outlook on further steps. 2. CHIP AND INSTRUMENT The developed automated sample preparation instrument weighs around 8 kg with a length of 300 mm, a depth of 250 mm and a height of 150 mm. Two custom made syringe pumps allow for the transport of all liquids inside the chip system (see Figure 1).

Smart Biomedical and Physiological Sensor Technology IX, edited by Brian M. Cullum, Eric S. McLamore, Proc. of SPIE Vol. 8367, 83670G · © 2012 SPIE · CCC code: 1605-7422/12/$18 · doi: 10.1117/12.923652

Proc. of SPIE Vol. 8367 83670G-1

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Underneath each of the six turning valve sits a stepper motor (Dr. Fritz Faulhaber GmbH, Schönaich, Germany) with a allow for the automatic rotation and positioning of each valve. To get the home position of each motor, a reflective light barrier is mounted with the motor, thus making it possible to calibrate the motor and to adjust fabrication tolerances.

The whole sample preparation unit is powered via a 24V DC power supply unit and controlled via an embedded microcontroller board (Analog Devices GmbH, Munich, Germany) therefore allowing a fully automated assay after the sample is manually inserted and the start button is pressed.

The disposable LOC system was made of the polymer COP 1420R (Zeon Europe GmbH, Düsseldorf, Germany) which is transparent down to the ultra violet spectrum and exhibits low water absorption. The chip was produced by injection moulding[7] and contained several fluidic channels, reservoirs for lysis, wash and extraction buffers and chambers for a sterile filter and a silica matrix. It also contained four luer adapters that served as liquid inlets or outlets. The outlets transferred the eluate into the reaction vessel, and excessive sample into an external waste reservoir. The chip further included different through holes compatible with so-called turning valves, these valves being separately manufactured by injection moulding. The chip had a dimension of 133x64x3mm³ (see Figure 3).

Figure 3. Picture of an assembled injection-moulded chip with valves, mixing and filter elements and a silica membrane.

After the injection moulding, manual assembly was required to integrate a filter (pore size of 0.2 µm), the silica

matrix, two mixers and six turning valves. Subsequently a silica matrix disc (Molzym GmbH, Bremen, Germany) with a diameter of 8 mm was pressed into a chamber of the chip. This sliced ring held down the silica matrix while leaving its middle open for flow-through (see Figure 4). Two Kenics mixers (GLT GmbH, Pforzheim, Germany) were inserted into two separate chambers of the chip, allowing for the homogeneous mixing of the sample with different buffers in a split-and-recombine approach (see Figure 4).

Figure 4. Assembled silica matrix, pressed down with a clamp ring made from polypropylene (left); Kenics mixer with seven mixing

elements made of polypropylene (right).



The chip was sealed by pressing an adhesive foil (Abgene Ltd., Epsom, UK) onto the chip. This foil left open only

the four luer adapters for inserting and extracting the sample, the through holes for the valves and 12 holes for filling the buffers into the chip reservoirs before testing (two filling holes for each buffer).

At the end of the assembly process, six turning valves were connected with the chip by pressing each of them onto two pins. Each valve consisted of a polymer housing and a rotor made of polyetherketone (Ensinger GmbH, Nufringen, Germany) rotated via a coupling by a driver motor. A fluoro-elastomeric seal disc (Rala GmbH, Ludwigshafen, Germany) is glued onto the opposing side of the rotor with instant adhesive. The seal contains cavities with a depth of 0.4 mm and a width of 0.8 mm, manufactured by laser ablation (see Figure 5).

Figure 5. Schematic cross section of a turning valve (left); top view of the assemble valve showing the green seal with its laser ablated

cavities (right).

By assembling the valve onto the chip, the flouro-elastomer is pressed onto the chip surface via a spring, thus sealing the area surrounding the through holes of the chip. Any two through holes of the chip can be connected by a seal cavity after turning the rotor while other through holes are closed simultaneously. Before an assay was started, all valves were automatically turned into their start position by the instrument. Additionally, all light barriers were checked for proper functioning. 3. EXPERIMENTAL PREPARATION AND BIOLOGICAL VALIDATION After mounting the valves, the LOC on-chip reservoirs were filled via pipette with buffers used for bacteria lysis and DNA extraction which were taken from a commercially available laboratory kit (Presto-Spin-D-Kit, Molzym GmbH, Bremen, Germany). The kit comprised 250 µl of an aqueous lysis buffer (buffer EX) containing salts and detergents for heat-induced destruction of the bacterial cell wall, 50 µl of a stabilizer buffer (buffer CH), 240 µl of a high salt incubation buffer (buffer AB) for DNA binding onto the silica matrix. The chip was furthermore filled with two washing buffers each with a volume of 330 µl (buffer WB and 70% EtOH). The chip was then filled with 130 µl of an elution buffer (buffer EB), to extract the cleaned DNA from the silica matrix. The position of the buffer reservoirs can be seen in Figure 6.



Figure 6. Buffer reservoirs on the sample preparation chip.

After filling each buffer reservoir, the filling holes were closed by inserting a stainless steel pin into each of them.

During the assay the buffer reservoirs were accessible via the turning valves whereas the turning valves were closed during filling (see Figure 7).

Figure 7. Buffer storage reservoir on chip (left), schematic filling of the reservoir with mounted turning valve (right).

A 15 ml plastic tube was placed into the instrument below the waste exits of the chip as a waste reservoir for

excessive liquids. Afterwards, a needle was mounted onto the outlet luer adapter of the chip through which the eluted DNA was dispensed into a reaction vessel below the chip after the sample preparation assay was completed. Finally, the chip was set into the instrument and locked into its position by pressing a lid onto it.

9 ml of a liquid sample loaded with bacteria and an excess of 1 ml air was drawn into a disposable polymer syringe which was inserted into PUMP 1, then connected with the chip inlet via silicone tubing and the bacteria suspension was pumped into the chip system. The germs were concentrated on the surface of the sterile filter while the excessive sample volume passed over the filter and dropped into the waste reservoir.

Afterwards, the valves were turned and the lysis buffer EX was pumped out of its storage reservoir, back-flushing the bacteria from the filter (using PUMP 2). Since the capillary forces of this filter are quite high due to its small pore size it was not recommendable to pump air through it once it was wetted since this lead to a large pressure increase inside the chip system with the risk of breaking the filter material. To avoid this, the lyses buffer EX was pumped until the end of the plug was above the filter. Now, the succeeding air cannot cross the filter without increasing the pump pressure to more than 2.5 bar. This pressure rise was detected by the pressure sensor inside the device which hence stopped PUMP 2 before the pressure was above 1 bar. The first two turning valves were rotated and the remaining air inside the sample syringe (using PUMP 1) was used to mix buffer EX (containing the filtered bacteria) with the stabilization buffer CH. This approach lead to a small loss of lysed sample but circumvented the pumping of air above the filter and the risk of destroying it meanwhile. The pressure to transport the liquids through the rest of the system can therefore be reduced to a maximum of 0.25 bar.



The mixture of the two buffers EX and CH was transported through the first Kenics mixer, guaranteeing a thorough and even mixture of the two liquids. After the liquid plug arrived at the first light barrier, the signal loss can be detected and was used to switch off PUMP 1 stopping the whole liquid plug in the channel situated above the first heater.

The two turning valves before and after the heater were rotated to confine the liquid there during heating enabling the minimisation of vaporisation during thermo-chemical lysis. For 10 minutes the sample was heated up to 93°C, guaranteeing the break up of the bacteria cell walls and the release of genomic DNA.

After lysis, both turning valves were opened again, and the sample was transported until it arrived at the next light barrier. Then, the next turning valve was rotated and the incubation buffer AB was pumped out of its storage reservoir. Mixing with the sample was performed by pumping the mixture through a second Kenics mixer, leading to sufficient high salt concentration which is necessary for DNA binding on any silica surface. The whole sample plug (now 540 µl in sum) was finally pumped through the silica matrix where the DNA was bound to the matrix and the excessive liquid was pumped into the waste reservoir. Unwanted contaminations like cell fragments and proteins were removed by subsequently flushing the silica membrane with the two washing buffers, using PUMP 2.

Since the alcoholic washing puffers inhibit subsequent PCR steps, all residua must be removed. This was ensured by blowing air for several minutes through the membrane while heating it up to 70°C. Afterwards, 130 µl elution buffer EB was pumped through the silica membrane and incubated there for several minutes. Then, the elution buffer was pumped further on and transferred into the reaction vessel. The DNA eluate inside the reaction vessel can then be manually gathered and be mixed with different reagents for subsequent analysis, like e.g. PCR master mixes and inserted into standard laboratory instruments. The whole sample preparation process including the concentration and the purification of a 9 ml sample spiked with bacteria down to a 130 µl DNA eluate is done totally automated within one hour. At the end, the chip must be removed together with the waste reservoir and the instrument is ready for the next run. 4. RESULTS A series of experiments were performed using the lab-on-a-chip system in the automated instrument indicating the reproducibility and reliability of the system. It was possible to extract bacterial DNA out of 9 ml liquid samples spiked with Escherichia coli. The DNA which was obtained by running the biological assay on the Lab-on-a-Chip system was analysed by standard Real-Time PCR using E.coli specific primers for a 113bp dxs gene product (forward primer: 5’-CGAGAAACTGGCGATCCTTA-3’, reverse primer: 5’- CTTCATCAAGCGGTTTCACA-3’)[8].

Figure 8 shows the results of a DNA extraction series, performed with 9ml water samples spiked with 104, 105, 106 and 107 E.coli bacteria. All tests were performed using the automated instrument whereas the Real-Time PCR was done after transferring the on-chip extracted DNA to a standard BioRad iQ5 cycler (Bio-Rad Laboratories GmbH, Munich, Germany). All results show sufficient amounts of DNA for amplification, indicated by cycle threshold (Ct) values of around 22 for 107 E.coli up to Ct values of around 30 for 104 E.coli. All sample preparation steps were completely performed with the automated system while the PCR was done in an iQ5 Real-Time Cycler by BioRad (SYBR Green Supermix; Primers dxs for. & rev.).



Figure 8. Real-Time PCR results showing the amplification of on-chip extracted DNA from 104, 105, 106, and 107 E.coli bacteria

spiked into 9 ml water.

Figure 9 shows the results of experiments which were performed with lower bacteria numbers. An amplification of on-chip extracted DNA was possible only with bacteria counts in the range of 104 and higher. On-chip experiments performed with a bacteria numbers of 102 and 103 showed negative results, most probably due to DNA losses on the sterile filter and the silica matrix.

Figure 9. Real-Time PCR results obtained from on-chip extracted E.coli DNA. 102, 103, 104, and 105 E.coli bacteria spiked into 9 ml

water.

Figure 10 shows graphs of further experiments comparing the results of E.coli DNA which was extracted and purified with the automated chip system compared to extractions done with the manual lab protocol using Molzym’s PrestoSpin D Plant Kit. For these experiments, liquid samples (9ml for the lab-on-a-chip-system respectively 1 ml for the standard lab protocol) were spiked with 105, 106 respectively 107 E.coli bacteria. The amplification graphs show that the



DNA extraction and purification done with the automated lab-on-a-chip system is comparable to the results gained with the standard spin tube approach, at least for higher bacteria numbers. The respective graphs for both 107 and 107-Standard and 106 and 106-Standard are almost identical, while the 105-Standard graph ascends earlier than the 105graph obtained from on-chip extracted DNA.

Figure 10. Diagram comparing the results of E.coli DNA extracted with the automated chip system to extractions done with the standard lab protocol using spin tubes (Molzym PrestoSpin D Plant Kit). DNA extraction and purification was done with liquid samples, spiked with 105, 106 respectively 107 E. coli. DNA extractions which were done with the Molzym Kit are marked “105-

Standard, 106-Standard, 107-Standard” while the chip results are marked “105, 106, 107”.

This demonstrates that the extraction of genomic DNA from higher bacteria counts with the chip system is equal to the standard lab method while the performance of the chip system for smaller bacteria numbers is lower than the manual Molzym standard, for which the limit of detection goes down to 103 bacteria, whereas the limit of detection for the sample preparation chip is 104 (which can be seen in Figure 9). This is most likely due to DNA losses on the sterile filter. Furthermore, it was found out that small residues of EtOH still left in the elution buffer from the previous washing steps, are inhibiting the subsequent PCR, shifting the Ct values to higher cycle numbers. 5. CONCLUSIONS After showing the successful amplification and detection of on-chip extracted bacteria DNA, next steps will be the ongoing optimisation of the biological assay and a further evaluation of different sterile filters and silica membranes which might be even better suited for a lab-on-a-chip system. This could improve sensitivity and might lead to the detection of even lower bacteria numbers. Additionally, the automated lab-on-a-chip system should be evaluated with different types of bacteria, e.g. Bacillus thuringiensis, Bacillus Anthracis or Yersinia pestis. Furthermore, a more thorough drying procedure after the two washing steps of the DNA bound onto the silica matrix is necessary, removing the remaining residues of EtOH in the subsequent elution step.

The next integration steps will be the implementation of additional functionalities into the system. This can be the further integration of sample preparation methods, e.g. for the extraction of bacteria out of soil samples, the integration of an on-chip PCR for the amplification of the eluted DNA and the implementation of an interface to other instruments, e.g. a commercial toxin detection system or a lateral flow dipstick for the verification of harmful germs where the filtrate can be further processed and the result can be displayed.

The whole manufacturing process of the LOC device is already highly compatible to mass production methods, starting with the production of the chip and the turning valves by injection moulding up to the simple assembly process which could easily be integrated into an automatic assembly line.



The instrument allows for the automated and fast generation of bacterial DNA within one hour, eliminating error-prone and laborious manual steps of highly trained personnel. The device weighs only around 8 kg and is therefore portable. It can be operated as a stand-alone device with the simple addition of batteries. This instrument therefore opens up the development of wholly integrated and portable sample preparation and detection systems for a broad application range. This could be on-spot food controls, point-of-care life sciences, or effective counterterrorism measures in the field. Acknowledgments This work was supported by Wehrwissenschaftliches Institut für Schutztechnologien -ABC-Schutz (WIS) (Humboldtstraße, 29633 Munster, E/E590/5Z013/4F169, E/E590/6Z012/4F169, E/E590/7Z012/4F169, E/E590/8Z016/4F169, E/E590/8Z016/7F225, and E/E590/9Z015/4F169). References [1] Yole Development Company, Market Study „BioMEMS“, 2008. [2] C. D. Chin, V. Linder, S. K. Sia, “Lab-on-a-chip devices for global health: Past studies and future opportunities”, Lab Chip, 2007, 7, 41–57. [3] B. Weigl, G. Domingo, P. LaBarre and J. Gerlach, “Towards non- and minimally instrumented, microfluidics-based diagnostic devices”, Lab Chip, 2008, 8, 1999 - 2014, DOI: 10.1039/b811314a. [4] C.J. Easley, J.M. Karlinsey, J.M. Bienvenue, L.A. Legendre, M.G. Roper, S.H. Feldman, M.A. Hughes, E.L. Hewlett, T.J. Merkel, J.P. Ferrance, and J.P. Landers, “A fully integrated microfluidic genetic analysis system with sample-in–answer-out capability”, PNAS , 2006, vol. 103, no. 51, 19273. [5] Y.-F. Chen, J. M. Yang, J.-Jr. Gau, Ch.-M. Ho, Y.-Ch.Tai, “Microfluidic System for Biological Agent Detection”; http://ho.seas.ucla.edu/publications/conference/2000. [6] I. Abdel-Hamid, A. Linnell, G. Bajszar and C. Call, “Portable Microfluidic Biological Agent Detection System”; http://handle.dtic.mil/100.2/ADA433114. [7] H. Becker , C. Gärtner, “Polymer microfabrication methods for microfluidic analytical applications”. Electrophoresis 2000 Jan; 21(1):12-26 [8] Lee C., Kim J., Shin S.G., Hwang S. (2006) Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli, Journal of Biotechnology 123:273–280



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