Overview

Algal bloom can happen depending on the environmental conditions, and it often has harmful effects to the ecological systems.  Therefore it is important to monitor the content of the sea water and assess the concentration of different algae.  Lab-on-chip offers several advantages.  The miniaturized device reduces the total sample needed and also decrease the total time needed for detection.  Also, the chips can be fabricated in large quantities with minimal cost so many experiments can be run in parallel.   Eventually, the chip can be integrated to an automated system that can be deployed to many different sites for sea water monitoring. 

Our project is organized into two main research areas.  The first one is a chip for separating different algae and count the number of algae with an impedance sensor.  The chip will first be tested with polystyrene beads and sea water sample in the lab.  The eventual goal is to make a stand-alone unit that can be deployed.  The second main area of this project is to make a chip that can culture algae and screen for factors inducing toxin production.  Pseudo-nitzschia is one type of algae that produces a neural toxin called Domoic Acid.  However, during Pseudo-nitzschia bloom, domoic acid is not always produced.  In another word, growth of algae does not equal domoic acid production.  Many factors (such as metal, nutrient, or ionic concentration) might induce or suppress algae to produce toxin.  Yet, exact causes are unclear.  To speed up the process of screening for possible factors inducing toxin production, we would like to make a chip that culture Pseudo-nitzschia under different growing conditions. 

Systems/Experiments

Separation based on particle sizes

The separator design involves a separation chamber filled by an array of cylindrical pillars.  Each row of pillars has the same center-to-center distance l and edge-to-edge distance d, and is staggered by a finite displacement from the previous row Dl (Figure 1B).  Because of small channel dimension (on the order of 10-100 μm) and flow rate (on the order of 0.01-1 μl/min), the Reynolds number is below 1.  Thus the flow inside the device can be treated as 2D laminar flow.  For fully developed steady flow, each pillar has two stagnation points.  The stagnation points of adjacent rows are rotated with respect to the net fluid flow direction because of the row shift.  Separation lanes are defined by division lines, which are streamlines ending at stagnation points (Figure 1C).  We also define the critical separation size as twice the minimum distance between the edge of pillar and the nearest division line.  For a given geometrical design, there is one corresponding critical separation size. 

Figure 1: (A) Device for particle separation. (B) Detailed device structure with geometrics labeled. (C) A small particle moves in zigzag mode and a large particle moves in displacement mode with four separation lanes for Dl=l/4

When the particles are not interacting with walls, we assume they will not change the flow pattern and will follow streamlines.  The interactions between particles are also neglected for simplicity of the model.  If the diameter of a particle is smaller than the critical separation size, it can follow a separation lane exactly resulting in a zigzag flow pattern which follows the net fluid flow direction over a long distance.  On the other hand, if the diameter of a particle is larger than the critical separation size, it flows in displacement mode, meaning it does not remain in one separation lane all the time, and changes between lanes as it is unable to make sudden turns around pillars.  These particles flow diagonally and do not follow the net fluid flow direction. 

We chose PDMS (Sylgard 184, Dow Corning, MI) for the device because of its ease of use. Devices are fabricated with DRIE-silicon molds and mounted on a glass slide by overnight baking at 80 °C (Figure 1A). The channel height is 20 μm. The effective separation area measures 7mm by 1.8 mm and consists of upstream and downstream regions. In each region, every consecutive row of obstacles is shifted horizontally by a fixed amount: 4 μm for upstream, 6 μm for downstream. In both regions, along each row, the center-to-center distance of obstacles λ is 60 μm while the distance between them, d, is 14 μm. At the inlet, sample is introduced and focused by two sheath flows.

Impedance sensing

After algae cells are roughly separated by size upstream, downstream impedance sensors are proposed to count and cell numbers and also further differentiate cell types.  Coulter sensors, which are impedance sensors in DC mode have been used as standard way to perform blood count.  The integration of signal magnitude in Coulter sensors is proportional to particle volume. 

Models for impedance response of mammalian cells in micro devices have been proposed.  Under low frequency, the impedance is dominated by double layer impedance of cell membrane.  For higher frequency, normally in MHz range, the internal structures of cells dominate.  So cells with different internal electrical properties can be distinguished.

Recently, T. Muller Th. Schnelle and G. Fuhr studied dielectric spectra of snow algae Chloromonas nivalis and Chlamydomonas nivalis by electrorotational measurement.  They found in this case the complex structured cell wall, which has low permittivity, dominate impedance under 1MHz.  At higher frequencies, the chloroplasts and cytoplasm, which is filled with small vacuoles and a large number of organelles, play more important roles.  Different models were proposed and fitted with experimental data.

In our design, we proposed to use Coulter sensors (DC mode) to count algae number and measure high frequency impedance to differentiate different algae types.

Algae culture on chip

Microfabrication technology will be used to make the chip.  The culture chamber and mixer will be made up of parylene because parylene is known to be a bio-compatible material.  Figure VIII-2 shows the schematic of the chip design.  One key aspect of the device is the combinatorial mixer that will use 3D microfluidic to mix the input streams into different compositions.  The cells will be kept inside the culturing chamber by the filter, and the combinatorial mixer will feed different media into each culture chamber.  This chip will allow multiple experiments to be run in parallel.  

Figure 2: Schematic chip design showing the different components that will be on the chip.

For the separator device, the devices are first calibrated with polystyrene beads.   Fluids can be pumped into devices with syringe pumps (Pico Plus, Harvard Apparatus, MA).  The images can be captured by a microscope equipped with CCD.  Finally, sea water containing different algae will be pumped into the device and be separated based on their sizes. 

For the algae culture on chip device, cells will be loaded into the chip through the cell loading port.   The media will be delivered into each culture chamber, and the growth of the cells inside the chip will be monitored with microscope.  The toxin will be detected and quantified using an immuno-based assay (ELISA). 

Accomplishments

For a preliminary study, a separator device is designed and fabricated.  Simulation based on described theory predicts the critical separation size to be 7.1 μm (Figure 3) while experiments measured value based on polystyrene beads is around 8 μm (Figure 4).

Figure 3: Simulated flow field inside device.

Figure4: Separation function curve.

We tested separation of four types of algae with the device.  As shown in Figure VIII-5, they have different diameters: Aureococcus anophagefferens, 2-3 μm; Chlorella stigmatophora, 3-6 μm; Heterosigma akashiwo, 10-15 μm; Chlamydomonas sp., 10-15 μm.  For current device, Aureococcus anophagefferens and Chlorella stigmatophora can be separated (Figure 6).  Aureococcus anophagefferens follows zigzag mode while Chlorella stigmatophora flows in displacement mode.  Heterosigma akashiwo and Chlamydomonas sp seem to be larger than the gap size, so they are captured inside device.

Figure 5: Four types of algae under bright light microscope.  (A) Aureococcus anophagefferens  (B) Chlorella stigmatophora (C) Heterosigma akashiwo  (D)Chlamydomonas sp.

Figure 6: Four types of algae tested in device.  (A) Aureococcus anophagefferens  (B) Chlorella stigmatophora (C) Heterosigma akashiwo  (D)Chlamydomonas sp.

The algae culture on chip device is in its initial phase.  The design of the chip has been drawn out, and the protocol for the fabrication process has been written. 

Future Directions

• Test the separation efficiency of the separator device.
• An impedance sensor will be designed and fabricated to count the number of cells.
• The impedance sensor will be integrated with the separator to make a complete unit. 
• The algae culture chip will be fabricated.
• Cells will be loaded onto the chip, and the growth/heath of the cells will be monitored.
• The toxin will be detected off-chip by ELISA.

People

Yu-Chong Tai, Professor, Caltech
Mike Liu, GSR, Caltech
Siyang Zheng, GSR, Caltech