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Individual cells immobilization for water-borne pathogen detection and enumeration
Procedia Chemistry Procedia Chemistry 1 (2009) 337–340 www.elsevier.com/locate/procedia
Proceedings of the Euro sensors XXIII conference
Individual cells immobilization for water-borne pathogen detection and enumeration 1
Qasem Ramadan1∗ and Lay Christophe1 Institute of Microelectronics, Agency for Science, Technology and Research, Singapore * Currently: Laboratory of Microsystems, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
Abstract
A cell counting device has been proposed and implemented for water-borne pathogen detection for drinking water quality monitoring applications. Our approach is based on magnetically-labelled cells immobilization in a high density array of individual cell for optical cell counting. The device has been tested for two water-borne pathogens: Giardia Lamblia & Cryptosporidium. An individual cell immobilization efficiency of 82% was achieved. Keyword:individual cell, pathogen, magnetic particles
1.
Introduction
The U.S Environmental Protection Agency (USEPA) has developed standardized method1623 for detection of Giardia & cryptosporidium cytes in water [1]. A major step of the standard protocol is cell counting which possesses an operational challenge due to the long processing time and the high possibility of errors. Therefore, it is highly desirable to perform such process with an automated system that minimize human interference and reduce possibilities of errors. Current cell quantification, particularly in biomedical fields, is performed using Fluorescenceactivated cell sorting (FACS) which make cell sorting affordable in many applications where samples contain a large number of cells. Several efforts have been made to developed micro-fluidic based cell counting devices [2-3] by integrating fluorescence detection into micro-fabricated chips. Beside the complicated fabrication processes required to develop these devices, they don not have the capability to provide accurate count of cells particularly when cells are diluted in large sample volume e.g. less than 1000 cells in 1 ml. Rodriguez et al [4] developed a prototype for counting of CD4 cells by capturing the stained cells on a membrane within a micro-chip followed by imaging the captured cells and converting the digital image into a cell count using computer algorithm. However, the captured cells are randomly distributed on the membrane which may results in cell random clustering. In the case of small number of cells in large sample volume, such as water-born pathogens, this may sharply reduce the counting accuracy. Therefore, there is still need to develop a method for cell immobilization in individual cell manner to facilitate the counting process that utilizes digital imaging methods. Cellular microarrays have been demonstrated hydro-dynamically employing micro-fabricated well arrays [5,6]. However, these approaches aimed to assemble cell array from a large population for single cell studies without a mechanism to capture all the targeted
∗
Corresponding author. Tel.: +41 21 693 6759; fax: +41 21 693 5950, E-mail address: [email protected]
1876-6196/09/$– See front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.proche.2009.07.084
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Q. Ramadan and L. Christophe / Procedia Chemistry 1 (2009) 337–340
cells. In this paper we describe a simple and robust micro-fluidic chip for cells immobilization that efficiently facilitate cell counting process and in the same time complies with the standard protocol. 2.
Concept and Methodology
Two magnetic based cell immobilization mechanisms have been designed and implemented: Fig.1 shows a schematic representation of these two approaches. The first mechanism employs magnetic post deposited inside a micro-well array (Fig. 1a). A steep magnetic potential well is created by exposing the post to a magnetic field using an external magnetic source which can be a permanent or electromagnet. Each µ-well has a diameter of either 5 or 12 µm which are close to the size of either Giardia & cryptosporidium, respectively so that can trap only a single cell. The flow rate of the introduced sample has been adjusted to push any possible cell crowding to the next well(s). The second mechanism employs the coupling between planar micro-scale current-carrying conductors (CCCs) and a permanent magnet to generate a programmable magnetic force on the cell-beads suspension (Fig. 1b). CCCs can be electrically functionalized to create attracting and rebelling zones according to the current direction of each loop. Hence the magnetic field direction can be either parallel/anti parallel to the external field direction and the magnetization of magnetic particles suspension. The simulated magnetic flux profiles are shown in Fig.1c and d for each trapping mechanism. Fig. 2 shows the fabrication processes for the two devices. In the first approach, NiFe magnetic posts were deposited in a previously defined micro-well array created in Benzo-Cyclo-Butene (BCB) on a silicon substrate. The NiFe posts have thickness of 2 -4 µm leaving 6-8 µm deep micro-wells. In the second approach, High aspect ratio trenches (10 µm deep and 3 µm wide) were etched in silicon, passivated with a thin (~1000 Å) oxide layer, filled by Cu electroplating and then surface planarized using Chemical Mechanical Polishing (CMP), respectively. A second passivation SiO2 film and an Al layer (each ~1 µm thick) were deposited and patterned, the latter forming the bonding pads. The final device in both cases was inserted in a PMMA holder to facilitate beads/cell injection, exposing the magnetic structure to an external magnetic field, and connecting the CCCs to an electric current source (Fig. 3).
(a) Micro-well array
(b) Current carrying conductors
(c) Magnetic flux density generated by magnetic post array (d) Magnetic flux density generated CCCs Figure 1: Schematic representation (a and b) and magnetic flux density profile (c and d) of the two single cell immobilization devices.
Q. Ramadan and L. Christophe / Procedia Chemistry 1 (2009) 337–340
Seed layer deposition
BCB deposition
BCB patterning
339
NiFe deposition
Deep trench etching
Oxide, barrier & seed Chemical mechanical Al deposition layers deposition and polishing Cu electroplating Figure 2: Fabrication processes for both the micro-well array (top) and CCCs (bottom) devices.
3.
Figure 3: Chip holder with the two chips in the insets above.
Results and Discussion
Fig.4a shows magnetic beads with diameter of 2.8 µm trapped in the well array with distribution of 2-4 beads/well while Fig.4b shows clusters of magnetic beads with diameter of 1 µm trapped in CCCs-trap zones with bead-clear in the adjacent rebelling zones. Fig.4c shows individual Giarda Lamblia cells captured in well array with no cell crowding observed and Fig. 4d shows individual magnetic beads with diameter of 10µm trapped by CCCs. The µwell array occupancy (filling fractionation) was increasing with time as shown in Fig.5. Fig.6 shows the capture efficiency of the two structures for spiked known numbers of magnetic beads (N=500), Giardia and Cryptosporidium cytes (N=100). Fig. 7 shows counting results of number of trapped cells in the trapping zones for the CCCs.
(a)
(b)
(c) (d) Figure 4: (a) Magnetic beads with diameter of 2.8 µm trapped in the well array with distribution of 2-4 beads/well, (b) Clusters of magnetic beads with diameter of 1 µm trapped in CCC-traps; (c) Single Giardia cytes trapped in µwell array and (d) single bead trapped in CCC-traps.
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Q. Ramadan and L. Christophe / Procedia Chemistry 1 (2009) 337–340
100
Filling Fraction (%)
80
60 Magnetic post array CCCs
40
20
0 0
1
2 3 Time (min)
4
5
Figure 5: Beads occupancy (filling fraction) increases with time for both magnetic post array and CCCs. 1.0
µ−Magnetic posts µ−CCCs
100
Fraction of traps
Capture Efficiency (%)
0.8 80
60
40
20
0.6
0.4
0.2
0 Beads (diameter=5 um)
Beads (diameter=10 um)
Giardai Cells (8-12 um) Cryptosporidium cells (2-4 um)
0.0 1
2
3
4
Number of trapped cells
Figure 6: Capture efficiency of µ-posts and µ-CCCs for magnetic beads, Giardia & Crypto cytes.
Figure 7:Number of trapped cells counts in the trapping zones for the CCCs.
4. Conclusions Individual bead & cell immobilization in arrays was demonstrated using two magnetic mechanisms with immobilization efficiency of 82%. The two mechanisms were compared. The realized devices provide an efficient tool for water-borne pathogen detection in an integrated system that complies with the standard protocol. References 1. 2. 3. 4. 5. 6.
Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA (http://www.epa.gov/microbes/1623de05.pdf). Y.N. Wang, Y. Kang, D. Xu, C. H. Chon, L. Barnett, S. A. Kalams, D. Li and D. Li. On-chip counting the number and the percentage of CD4+ T lymphocytes. Lab Chip, 2008, 8, 309-315. C. C. Lin, A. Chen & C. H. Lin. Microfluidic cell counter/sorter utilizing multiple particle tracing technique and optically switching approach. Biomed Microdevices, 2008, 10, 55–63. W. R. Rodriguez, N. Christodoulides, P. N. Floriano, S. Graham, S. Mohanty, M. Dixon, M. Hsiang, T. Peter, S. Zavahir, I. Thior, D. Romanovicz, B. Bernard, A. P. Goodey, B. D. Walker and J. T. McDevitt, PLoS Med., 2005, 2, e182. J. R. Rettig and A. Folch, Large-scale single-cell trapping and imaging using microwell arrays, Anal. Chem.2005, 77, 5628-5634. D. D. Carlo, N. Aghdam and L. P. Lee, Single-cell enzyme concentrations, kinetics, and inhabitation analysis using high-density hydrodynamic cell isolation arrays, Analytical Chemistry, 2006, 78(14) 4925-4930.
Proceedings of the Euro sensors XXIII conference
Individual cells immobilization for water-borne pathogen detection and enumeration 1
Qasem Ramadan1∗ and Lay Christophe1 Institute of Microelectronics, Agency for Science, Technology and Research, Singapore * Currently: Laboratory of Microsystems, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
Abstract
A cell counting device has been proposed and implemented for water-borne pathogen detection for drinking water quality monitoring applications. Our approach is based on magnetically-labelled cells immobilization in a high density array of individual cell for optical cell counting. The device has been tested for two water-borne pathogens: Giardia Lamblia & Cryptosporidium. An individual cell immobilization efficiency of 82% was achieved. Keyword:individual cell, pathogen, magnetic particles
1.
Introduction
The U.S Environmental Protection Agency (USEPA) has developed standardized method1623 for detection of Giardia & cryptosporidium cytes in water [1]. A major step of the standard protocol is cell counting which possesses an operational challenge due to the long processing time and the high possibility of errors. Therefore, it is highly desirable to perform such process with an automated system that minimize human interference and reduce possibilities of errors. Current cell quantification, particularly in biomedical fields, is performed using Fluorescenceactivated cell sorting (FACS) which make cell sorting affordable in many applications where samples contain a large number of cells. Several efforts have been made to developed micro-fluidic based cell counting devices [2-3] by integrating fluorescence detection into micro-fabricated chips. Beside the complicated fabrication processes required to develop these devices, they don not have the capability to provide accurate count of cells particularly when cells are diluted in large sample volume e.g. less than 1000 cells in 1 ml. Rodriguez et al [4] developed a prototype for counting of CD4 cells by capturing the stained cells on a membrane within a micro-chip followed by imaging the captured cells and converting the digital image into a cell count using computer algorithm. However, the captured cells are randomly distributed on the membrane which may results in cell random clustering. In the case of small number of cells in large sample volume, such as water-born pathogens, this may sharply reduce the counting accuracy. Therefore, there is still need to develop a method for cell immobilization in individual cell manner to facilitate the counting process that utilizes digital imaging methods. Cellular microarrays have been demonstrated hydro-dynamically employing micro-fabricated well arrays [5,6]. However, these approaches aimed to assemble cell array from a large population for single cell studies without a mechanism to capture all the targeted
∗
Corresponding author. Tel.: +41 21 693 6759; fax: +41 21 693 5950, E-mail address: [email protected]
1876-6196/09/$– See front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.proche.2009.07.084
338
Q. Ramadan and L. Christophe / Procedia Chemistry 1 (2009) 337–340
cells. In this paper we describe a simple and robust micro-fluidic chip for cells immobilization that efficiently facilitate cell counting process and in the same time complies with the standard protocol. 2.
Concept and Methodology
Two magnetic based cell immobilization mechanisms have been designed and implemented: Fig.1 shows a schematic representation of these two approaches. The first mechanism employs magnetic post deposited inside a micro-well array (Fig. 1a). A steep magnetic potential well is created by exposing the post to a magnetic field using an external magnetic source which can be a permanent or electromagnet. Each µ-well has a diameter of either 5 or 12 µm which are close to the size of either Giardia & cryptosporidium, respectively so that can trap only a single cell. The flow rate of the introduced sample has been adjusted to push any possible cell crowding to the next well(s). The second mechanism employs the coupling between planar micro-scale current-carrying conductors (CCCs) and a permanent magnet to generate a programmable magnetic force on the cell-beads suspension (Fig. 1b). CCCs can be electrically functionalized to create attracting and rebelling zones according to the current direction of each loop. Hence the magnetic field direction can be either parallel/anti parallel to the external field direction and the magnetization of magnetic particles suspension. The simulated magnetic flux profiles are shown in Fig.1c and d for each trapping mechanism. Fig. 2 shows the fabrication processes for the two devices. In the first approach, NiFe magnetic posts were deposited in a previously defined micro-well array created in Benzo-Cyclo-Butene (BCB) on a silicon substrate. The NiFe posts have thickness of 2 -4 µm leaving 6-8 µm deep micro-wells. In the second approach, High aspect ratio trenches (10 µm deep and 3 µm wide) were etched in silicon, passivated with a thin (~1000 Å) oxide layer, filled by Cu electroplating and then surface planarized using Chemical Mechanical Polishing (CMP), respectively. A second passivation SiO2 film and an Al layer (each ~1 µm thick) were deposited and patterned, the latter forming the bonding pads. The final device in both cases was inserted in a PMMA holder to facilitate beads/cell injection, exposing the magnetic structure to an external magnetic field, and connecting the CCCs to an electric current source (Fig. 3).
(a) Micro-well array
(b) Current carrying conductors
(c) Magnetic flux density generated by magnetic post array (d) Magnetic flux density generated CCCs Figure 1: Schematic representation (a and b) and magnetic flux density profile (c and d) of the two single cell immobilization devices.
Q. Ramadan and L. Christophe / Procedia Chemistry 1 (2009) 337–340
Seed layer deposition
BCB deposition
BCB patterning
339
NiFe deposition
Deep trench etching
Oxide, barrier & seed Chemical mechanical Al deposition layers deposition and polishing Cu electroplating Figure 2: Fabrication processes for both the micro-well array (top) and CCCs (bottom) devices.
3.
Figure 3: Chip holder with the two chips in the insets above.
Results and Discussion
Fig.4a shows magnetic beads with diameter of 2.8 µm trapped in the well array with distribution of 2-4 beads/well while Fig.4b shows clusters of magnetic beads with diameter of 1 µm trapped in CCCs-trap zones with bead-clear in the adjacent rebelling zones. Fig.4c shows individual Giarda Lamblia cells captured in well array with no cell crowding observed and Fig. 4d shows individual magnetic beads with diameter of 10µm trapped by CCCs. The µwell array occupancy (filling fractionation) was increasing with time as shown in Fig.5. Fig.6 shows the capture efficiency of the two structures for spiked known numbers of magnetic beads (N=500), Giardia and Cryptosporidium cytes (N=100). Fig. 7 shows counting results of number of trapped cells in the trapping zones for the CCCs.
(a)
(b)
(c) (d) Figure 4: (a) Magnetic beads with diameter of 2.8 µm trapped in the well array with distribution of 2-4 beads/well, (b) Clusters of magnetic beads with diameter of 1 µm trapped in CCC-traps; (c) Single Giardia cytes trapped in µwell array and (d) single bead trapped in CCC-traps.
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Q. Ramadan and L. Christophe / Procedia Chemistry 1 (2009) 337–340
100
Filling Fraction (%)
80
60 Magnetic post array CCCs
40
20
0 0
1
2 3 Time (min)
4
5
Figure 5: Beads occupancy (filling fraction) increases with time for both magnetic post array and CCCs. 1.0
µ−Magnetic posts µ−CCCs
100
Fraction of traps
Capture Efficiency (%)
0.8 80
60
40
20
0.6
0.4
0.2
0 Beads (diameter=5 um)
Beads (diameter=10 um)
Giardai Cells (8-12 um) Cryptosporidium cells (2-4 um)
0.0 1
2
3
4
Number of trapped cells
Figure 6: Capture efficiency of µ-posts and µ-CCCs for magnetic beads, Giardia & Crypto cytes.
Figure 7:Number of trapped cells counts in the trapping zones for the CCCs.
4. Conclusions Individual bead & cell immobilization in arrays was demonstrated using two magnetic mechanisms with immobilization efficiency of 82%. The two mechanisms were compared. The realized devices provide an efficient tool for water-borne pathogen detection in an integrated system that complies with the standard protocol. References 1. 2. 3. 4. 5. 6.
Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA (http://www.epa.gov/microbes/1623de05.pdf). Y.N. Wang, Y. Kang, D. Xu, C. H. Chon, L. Barnett, S. A. Kalams, D. Li and D. Li. On-chip counting the number and the percentage of CD4+ T lymphocytes. Lab Chip, 2008, 8, 309-315. C. C. Lin, A. Chen & C. H. Lin. Microfluidic cell counter/sorter utilizing multiple particle tracing technique and optically switching approach. Biomed Microdevices, 2008, 10, 55–63. W. R. Rodriguez, N. Christodoulides, P. N. Floriano, S. Graham, S. Mohanty, M. Dixon, M. Hsiang, T. Peter, S. Zavahir, I. Thior, D. Romanovicz, B. Bernard, A. P. Goodey, B. D. Walker and J. T. McDevitt, PLoS Med., 2005, 2, e182. J. R. Rettig and A. Folch, Large-scale single-cell trapping and imaging using microwell arrays, Anal. Chem.2005, 77, 5628-5634. D. D. Carlo, N. Aghdam and L. P. Lee, Single-cell enzyme concentrations, kinetics, and inhabitation analysis using high-density hydrodynamic cell isolation arrays, Analytical Chemistry, 2006, 78(14) 4925-4930.