TOWARDS POINT-OF-CARE DIAGNOSTICS: A MICROFLUIDIC SAMPLE PREPARATION CHIP

FOR CONCENTRATION OF BACTERIA AND RNA EXTRACTION H. Hubbe, S. Hakenberg, G. Dame and G.A. Urban

Laboratory for Sensors, Department of Microsystems Engineering - IMTEK, University of Freiburg, Georges-Köhler-Allee 103, 79110 Freiburg, GERMANY

ABSTRACT

We report on the development and proof-of-concept of a microfluidic chip. It provides a full range of pretreatment steps for the point-of-care detection of bacteria with a total process time of less than 15 minutes. The device integrates concentration, lysis and nucleic acid purification. Successful operation is demonstrated using E. coli as a model organ-ism.

KEYWORDS

Sample pretreatment, bacteria concentration, RNA extraction, point-of-care diagnostic

INTRODUCTION Bioanalytics is a field of highly advanced kits and chemicals. Tedious manual work is involved, performing and

preparing the processes and peripheral lab equipment is needed. The idea of a micro total analysis system (µTAS) or Lab-on-a-chip system is to provide a platform reducing this effort. In order to compete with the latest standard kits, these systems have to be faster, cheaper, more reliable, automated or meet at least one of the attributes mentioned. [1]

Most bioanalytical processes can be divided into two main steps: Sample pretreatment and detection or processing. The focus of this work lies on an automatable integration of sample pretreatment into a microfluidic device. In a future combination with a suitable method of detection, this device is intended to provide a point-of-care diagnostic tool.

Figure 1: Lab-on-a-Chip for integrated concentration, lysis and nucleic acid purification; labels corresponding with

fig. 2. We present a microfluidic chip system that provides a full range of pretreatment steps for the detection of bacteria

with a total process time of less than 15 minutes (fig. 1). It is an integration of a previously presented concentration chip [2] into a lysis and nucleic acid purification chip [3,4]. The system combines unspecific free-flow electrophoresis with thermo-electrical lysis and nucleic acid purification.

FABRICATION

The device is manufactured on a 500 µm thick glass substrate (Pyrex). Fluidic structures are patterned by lamination and photolithographical structuring of several layers of a dry film resist (Ordyl, Elga Europe) as described by Vulto [5]. A second glass substrate bonded on top of the fluidic structures encloses the chip on the top side. The microfluidic chip is divided into two chambers by a hydrogel located in between. The two chambers have integrated multifunctional elec-trodes. They are deposited by evaporation onto the glass substrate and structured with a lift-off process. Complete and bubble-free priming and emptying of the chambers and the initial creation of the hydrogel barrier is assured by use of phaseguides as presented in prior work [6]. The structure of the phaseguides has been altered to a new schematic, show-ing a more reliable behavior in filling and refilling. Figure 2 depicts a diagram of the chip.

THEORY

A simplified schematic of the chip is shown in figure 3. The first step of the sample preparation in this device is a concentration of the bacteria. This enhances the detection rate of low concentrated samples. The compartment (A) in figure 2 is the “concentration chamber”. The sample enters this chamber, while an electric field is applied between the

A B Gel

978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 1641 17th International Conference on MiniaturizedSystems for Chemistry and Life Sciences27-31 October 2013, Freiburg, Germany

concentration and the elution electrode. Bacteria carrying the appropriate membrane charge for the chosen voltage polar-ity are being dragged along the direction of the electric field towards the hydrogel. They accumulate on the surface of the gel, having a diameter too large to enter the small pores of the gel. The sample fluid exits the chip and the continuous flow and accumulation leads to a rising bacteria concentration inside the concentration chamber.

The second step in sample preparation is the lysis of the collected bacteria. It follows immediately after the concen-tration. A high frequency (20 kHz) AC voltage of up to 200 V is connected to the concentration and the lysis electrode. The bacteria are lysed through thermoelectric effects.

In the last step, the RNA contained in the lysate is separated from interfering substances like cell debris and RNases and transferred to a buffer appropriate for the desired subsequent diagnostic processes (e.g. TE for RT-PCR). This buffer is previously added to the elution chamber (B). As an advantage of this device, there is again no manual intervention or mechanical actuation necessary. The purification can start immediately after the lysis, lowering the risk of sample deg-radation and complexity of the process. An electric field is applied between the concentration and the elution electrode, creating an electrical drag force on the negatively charged RNA molecules. After migrating through the hydrogel, the RNA can be collected from the elution chamber.

Figure 2: Simplified model of the chip system and experimental procedures.

EXPERIMENTAL

Experiments are conducted with E. coli as a model organism. The bacteria are prepared as over-night cultures in 10 ml LB-broth. For the experiments, 10 µl of the over-night culture are inoculated in 10 ml LB and incubated for two hours. This procedure guarantees a high amount of living cells.

The gel compartment of the chip is filled with a polyacrylamide gel and the elution chamber with a TBE buffer solu-tion. A syringe pump pumps the sample through the concentration compartment.

Experimental parameters are shown in table 1. The concentration time is varied between 3 and 9 minutes. The elu-ate is tested for RNA using real-time RT-PCR in a LightCycler system (Roche Diagnostics).

Table 1: Process times and additional parameters used for testing the chip.

Process step Time Parameters

Concentration 180-540 s flow rate 10 µl min-1

Ie = 300 µA

Lysis 180 s Upp = 200 V (sinusoidal) f = 20 kHz

Purification 300 s Ip = 120 µA Total 11-17 min

RESULTS

The results show lower ct values for increased concentration durations. Whereas 3 min of concentration and sample flow result in a ct value of 27, the 6 min process enhances the signal to a ct of 24. Additional concentration time (9 min) improves the value to a mean of 19. This result coincides with the expectations: Longer concentration times yield more bacteria, as more sample is processed and more bacteria accumulate on the hydrogel interface. This shows the function-ality of the on-chip concentration process.

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Figure 3: ct values of real-time RT-PCR performed in a LightCycler. The sample was divided and processed in dif-

ferent chips for different concentration times.

CONCLUSION The results of the concentration time variation show a strong enhancement of the PCR signal when extending the

process time in the device. Further experiments will clarify the actual cell yield achieved. The simplicity in the integration of the combined processes is the advantage of this system. A single pump is the on-

ly actuation device required for the chip’s operation. The chip represents a milestone with regard to a future point-of-care diagnostic tool for independent pathogen detection.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge funding by the German Federal Ministry of Education and Research (BMBF) (projects IMRA FKZ: 031A094A and SONDE FKZ: 130113 N). REFERENCES [1] M. Jürgen, K. Roppert, P. Ertl., Microfluidic Systems for Pathogen Sensing: A Review, Sensors, vol. 9(6) pp. 4804-

823, (2009). [2] S. Podszun, P. Vulto, H. Heinz, S. Hakenberg, C. Hermann, T. Hankemeier, G.A. Urban, Enrichment of viable bac-

teria in a micro-volume by free-flow electrophoresis, Lab on a chip, vol. 12(3), pp. 451–7, (2012). [3] P. Vulto, A Lab-on-a-Chip for automated RNA extraction from bacteria, Dissertation, Albert-Ludwigs-Universität

Freiburg, 2008. [4] P. Vulto, G. Dame, U. Maier, S. Makohliso, S. Podszun, P. Zahn, G.A. Urban, A microfluidic approach for high ef-

ficiency extraction of low molecular weight RNA, Lab on a chip, vol. 10(5), pp. 610–6, (2010). [5] P. Vulto, G. Medoro, L. Altomare, G. A. Urban, M. Tartagni, R. Guerrieri, and N. Manaresi, Selective sample

recovery of DEP-separated cells and particles by phaseguide-controlled laminar flow, Journal of Micromechanics and Microengineering, vol. 16(9), pp. 1847–1853, (2006).

[6] P. Vulto, S. Podszun, P. Meyer, C. Hermann, A. Manz, G.A. Urban, Phaseguides: a paradigm shift in microfluidic priming and emptying, Lab on a Chip, vol. 11(9), pp. 1596-602, (2011).

CONTACT G. Dame, tel: +49-761-203 7267; dame@imtek.de

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