DEVELOPMENT OF A POINT-OF-CARE BOVINE LIVE STOCK HEALTH CONTROL SYSTEM USING ACOUSTOPHORESIS

C. Grenvall1, P. Augustsson1, J. Riis Folkenberg2 and T. Laurell1 1Lund University, SWEDEN and

2FOSS Analytical A/S, DENMARK ABSTRACT

A microfluidic chip for acoustophoretic raw milk sample preparation, eliminating the need for chemical pretreatment linked to somatic cell counting, was developed. This opens the route to an on-farm platform for live stock health status analysis in the dairy industry.

Our previous work has shown the benefits of using acoustophoresis for raw milk analysis [1]. This work presents a combination of acoustophoresis and Coulter Counting to provide a simple point-of-care diagnostic tool to measure somat-ic cell count (SCC) levels, which indicate udder health [2]. Shorter treatment response times using this system will mi-nimize health hazards for live stock and consumers. KEYWORDS: Acoustophoresis, Ultrasound, Milk, Sorting

INTRODUCTION

Acoustophoretic separation of complex bio-suspensions has long shown promise [3]. Several groups have reported successful culturing and high viability of cells during and after exposure to acoustic processing and intensities corres-ponding to those used here [4,5]. By combining previously reported benefits of acoustophoretic removal of lipids with simultaneous cell coulter counting, the aim of the current work is to develop a novel live stock point-of-care system for SCC measurements. This will facilitate rapid detection of bovine diseases and address concerns about transfers of antibi-otic residues and pathogens to humans through dairy products whilst minimizing dairy-waste at the same time. The need for such systems are especially apparent in countries with a growing dairy market, eg. China and India, where the rapid increase of dairy farms raises demands on health status control of live stock, not generally addressable by centralized quality control systems.

This paper presents successful acoustophoretic pretreatment of raw milk samples from farms with high SCC corres-ponding to cattle with developed mastitis. The original samples were analyzed by standard flow cytometry and compared to corresponding Coulter Counter data with and without acoustophoretic processing for lipid emulsion removal.

THEORY

The acoustophoresis working principle is based on the force induced on particles in the gradient between minimum and maximum pressure nodes in standing wave fields. Depending on particle and media density and compressibility it is possible to achieve binary separation of particles. A convenient way of predicting the behavior of the suspended particles is to calculate the acoustic contrast factor (ACF (ϕ), Eq. 1) to determine if particles that are to be separated have opposite sign ACF. Positive sign particles travel towards the maximum pressure nodes (normally just called nodes) while negative sign particles travel towards minimum pressure nodes (normally called antinodes).

By utilizing the inherent laminar flow profile characteristics of microchannels, it is possible to separate and extract different particle categories through different outlets in continuous flow. In this work somatic cells have a positive ACF and focus in nodes while lipid vesicles have a negative ACF and focus in antinodes.

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ρ0 : density of the suspending medium ρp : density of the particle c0 : sound velocity in the suspending medium cp : sound velocity in the particle

EXPERIMENTAL

The acoustic chip was fabricated according to previously reported techniques [1]. The complete setup can be seen in fig 1 along with a schematic view of the chip with flows and acoustic profiles in fig 2a-c. Samples with 5.8*10^6 cells/ml entered the chip through the center inlet (35µl/min) and were hydrodynamically laminated using mQ-water (420 µl/min). Lipid depleted samples were extracted through the center outlet (60 µl/min) while lipids exited through the side outlets (390 µl/min). The small pressure surplus decrease air bubble formation. Samples were run through the chip with and without the acoustic power activated and collected in 100 µl loops during five minutes.

978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS 127 14th International Conference onMiniaturized Systems for Chemistry and Life Sciences

3 - 7 October 2010, Groningen, The Netherlands

Figure 1: An acoustophoretic setup with a signal generator, an amplifier, two syringe pumps with a total of four sy-

ringes, two sample valves, a microscope and a microfluidic chip. The chip (better visible in figure 2) fits in the micro-scope using a custom made holder to make room for fluidic connections and the electric cable running to the amplifier,

has a piezoceramic transducer attached underneath and teflon tubes for fluidic access.

Figure 2: (a) The acoustic chip was fabricated by creating a 1125 µm wide microfluidic channel through wet-etching of silicon followed by bonding of a glass lid on top thus sealing the channel. Fluidic access holes were etched from the back side of the chip. The acoustic force was induced using a piezoelectric transducer activated at 1.95 MHz,

corresponding to 3/2 λ across the channel, which was attached underneath the chip via an aluminium heat sink. (b) Raw milk samples with 5.8*10^6 cells/ml entered the acoustophoretic chip through the center inlet (35µl/min) and were hy-drodynamically focused in the centre using mQ-water (420 µl/min). Lipid depleted samples were extracted through the center outlet (60 µl/min) while lipids exited through the side outlets (390 µl/min). (c) The 3/2 λ width across the channel

allows for cells to focus into the middle outlet while lipids are separated into the side outlets. The two nodes closest to the sides prevent lipid aggregation with subsequent clogging and erroneous flows.

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c

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RESULTS AND DISCUSSION The acoustophoretic removal of the lipid fraction clearly reveals the cell peak (7-10 µm) in the Coulter Counter data,

normally not visible in the raw milk sample due to lipid content with the same size clouding the measurement, Fig 3a-b. Re-sults were compared with flow cytometry data for the same samples in order to verify extraction of the somatic cells from the lipid background. When analyzing the number of particles per volume in the Coulter Counter “cell peak” the samples con-tained an average of 6.3*10^6 cells/ml. This seems to suggest an offset of ~0.5*10^6 cells/ml as compared to flow cytome-try data. The slightly elevated levels are most likely attributed to the overlap of a small fraction of ≥7 µm lipid vesicles re-maining in the cell fraction.

Figure 3: (a) When analysing the original sample using Coulter Counting, the cell-sized lipid particles (>5 µm in

size) obscure the somatic cells, preventing label free SCC analysis. (b) In the acoustophoretically treated sample large lipid particles were almost completely removed which reveals a "cell peak" in the Coulter Counter data which corre-

sponds well to the measured SCC levels using flow cytometry.

CONCLUSION The obtained data clearly demonstrates that acoustophoresis in combination with Coulter Counter analysis offers a direct

method for somatic cell counting, eliminating the need for chemical sample pretreatment. On-going work targets the integra-tion of the acoustophoresis module with a microchip Coulter Counter.

ACKNOWLEDGEMENTS

The authors would like to thank Vinnova, Carl Trygger Foundation, Vetenskapsrådet, Foundation for strategic re-search, Craafordska Stiftelsen, and Kungliga Fysiografiska Sällskapet i Lund for funding. REFERENCES [1] C. Grenvall et al, “Harmonic Microchip Acoustophoresis: A Route to Online Raw Milk Precondition in Protein and

Lipid Content Quality Control” Anal. Chem., 81(15), 6195, (2009). [2] Y. H. Schukken et al, “Monitoring udder health and milk quality using somatic cell counts” Vet. Res., 34, 579,

(2003). [3] F. T. Petersson et al, Free flow acoustophoresis (FFA) – A new microfluidic based mode of particle and cell separa-

tion” Anal. Chem., 79(14), 5117, (2007) [4] J. Hultström et al, “Proliferation and viability of adherent cells manipulated by standing-wave ultrasound in a micro-

fluidic chip” Ultrasound Med. Biol., 33, 145, (2007) [5] M. Evander et al, “Non-invasive acoustic cell trapping in a microfluidic perfusion system for on-line bioassays”

Analytical Chemistry, 79, 2984, (2007) CONTACT Thomas Laurell, tel: +46-46-222 75 40; Thomas.Laurell@elmat.lth.se

Lipid content obscures cells

Cell peak clearly visible

b a Untreated sample

Acoustophoretically treated sample

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