Lead Investigator

Yu-Chong Tai, Caltech

Overview

Lab-on-Chip aquatic microorganism analysis system is a project that aims to expedite research in marine biology using chip-based technology. The miniaturized device reduces the total sample and detection time. Also, the chips can be fabricated in large quantities with minimal cost so many experiments can be run in parallel.   Our project is organized into two main research areas.  First, we would like to develop a chip to monitor the content of the sea water. 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.  The algal monitoring in sea water has not been automated and difficulties exist if one would like to have real-time distributed algal monitoring in sea water. It is proposed to developed lab-on-a-chip technology to monitor algal count of certain target specie.  The chip will take in sea water sample, separate the cells based on size, and a downstream impedance sensor will count the number of cells. 

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.

Approach

Chips will be fabricated using microfabrication technology.  The separator chip is designed to separate particles by size, and the particles can be counted using an impedance sensing chip.  The cell culture chip is composed of an array of culture chamber with an integrated combinatorial mixer.  The combinatorial mixer involves fabricating 3-dimensional fluidic networks and can be fabricated readily using our developed surface micromaching of parylene C. 

Systems/Experiments

1.  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 λ and edge-to-edge distance d, and is staggered by a finite displacement from the previous row Δλ (Fig. 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 having 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 (Fig. 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.

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.

 

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 Δλ=λ/4

 

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

We tested separation of four types of algae with the device.  They have the 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 (Fig 2).  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 2. Four types of algae tested in device.  (A) Aureococcus anophagefferens  (B) Chlorella stigmatophora  (C) Heterosigma akashiwo  (D)Chlamydomonas sp.

 

2.  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 counting of various particles such as blood cells.  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 (Fig 3, [1]).  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.

Figure 3. Models for impedance response of mammalian cells in micro devices [1].

 

We have completed the fabrication of the device by bonding PDMS defined channels to glasswith Ti/Pt patterned electrodes.  The design layout of this device is shown in Fig. 4.  The cells are injected into the inlet and pass through the aperture.  As each cell passes through, the impedance across the aperture changes and this result in the spikes.

 

Figure 4.  Design layout of the impedance sensor device.

 

The testing system and data acquisition system has also been completely built.  We have also tested the chip with polystyrene particles.  Fig. 5 demonstrated the chip can successfully count individual particles passing through the aperture.

Figure 5.  Detection of particles passing through the aperture.  As each particle passes through, the impedance across the aperture changes and this result in the spikes.

 

3.  Algae culture on chip for identifying toxin-production conditions

A chip to culture algae, Pseudo-nitzschia, under different conditions will be made to speed up the process for screening for the factors that induce toxin production.  The culture chamber and mixer will be made up of parylene because parylene is known to be a bio-compatible and chemically-inert material.  Fig. 6 shows the schematic of the chip design.  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).  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.  Many factors (such as metal, nutrient, or ionic concentration) might induce or suppress algae to produce toxin, and those factors will be tested with the culture algae.  Moreover, the combinatorial effect of different possible factors can be tested simultaneously.  Another advantage of this device is that this chip will allow multiple experiments to be run in parallel.

 

Figure 6.  Design of the algae culture chip showing the essential components.  The combinatorial mixer will take in the three inputs and output all possible combinations.

 

The device is monolithically fabricated with parylene C as the structural material.  The device has two microfluidic levels, and the 5-mask fabrication process is shown in Fig. 7.   The first-level channels were initially deposited, followed by the deposition of the overpasses and culture chambers.  The device was fabricated using our developed parylene surface micromaching technology: alternating layers of parylene C and sacrificial photoresist are deposited, and the sacrificial photoresist between two layers of parylene defines the channel.  After the photoresist is removed, the remaining parylene structures with open spaces become the fluidic networks of the chip.  Parylene C is compatible with lithographic CMOS/MEMS fabrication process and has been used for constructing microfluidics, bioMEMS and micro-total-analysis systems (μTAS). Also, parylene C has been proven to be chemically inert, as it has been used in many implantable devices. The chemically inert property of parylene C is particularly important because any byproduct generated from the breakdown of the culture chamber structures can deleteriously affect the cell culture experiments.  In addition, parylene C is transparent in the visible range and cells can be easily observed with light microscopy.

Fig. 8(a) shows the fabricated 1 cm x 1 cm chip, and the method of packaging the device.  A simple packaging scheme was developed to make the fluidic connections with the chip as shown in Fig. 8(b).  A piece of cured PDMS with punched holes was aligned onto the chip.  The PDMS and the chip were clamped together by two pieces of transparent acrylic that were milled with a computer-numerical-controlled (CNC) machine.  The PDMS acted both as a gasket layer to provide proper sealing and also as a port to receive the tubes.  Syringes were connected with Teflon tubes, which were plugged into the holes of the piece of PDMS.  Depending on the operation of the device, the PDMS piece can have holes open at different places to either close or open certain access holes on the chip.

 

Figure 7.  The monolithic fabrication process for making the 3-D microfluidic networks. Inserts in (2) and (4) show how the overpass is made by first etching open the parylene and then joining the two etched regions with the 2nd sacrificial photoresist.

 

Figure 8.  (a) Fabricated 1 cm by 1 cm chip.	Figure 8.  (b) Schematic of device packaging.	Figure 8.  (c)  Photograph of the packaged device.

 

We also tested the combinatorial mixer of the chip using different colors of food coloring and Fig. 9 shows that the combinatorial mixer is able to take in the 3 fluid streams and output the 7 possible combinations to the culture chambers.  The food coloring entering the control channel remains unmixed.  We have also culture B35 rat neuroblastoma cells on chip.  Fig. 10 shows that the chip is able to sustain cell growth.  In the future, growing algal cells using the chip will be performed.

 

Figure 9.  Combinatorial mixer demonstration. (a) Assembled device with food coloring being injected.  Green (G), red (R) and blue (B) food colorings are injected into the combinatorial-mixer inlets, while the yellow (Y) food coloring is injected into the control channel.	Figure 9.  (b)  Combinatorial mixer operated at 10 µL/min.  The scale bar represents 1 mm.

 

Figure 10. Demonstration of cell culture inside parylene C micro culture chamber.  B35 rat neuroblastoma cells were loaded into the culture chambers and the cells were allowed to attach to the surface for 4 hours.  The cells were grown with continuous perfusion of culture media and pictures were taken 4 hours, 16 hours and 42 hours after cells were loaded.

Reference

1. S. Gawad, K. Cheung, U. Seger, A. Bertsch, P. Renaud, Lab on a Chip 4, 241 (2004)

Accomplishments

• Completed fabrication of the impedance sensor chip
• Built the data acquisition system for the impedance sensor.
• Used the impedance sensor the count polystyrene beads.
• Completed fabrication of the culture chip with integrated combinatorial mixer
• Finished fluidic packaging system of the culture chip.
• Demonstrated mixing with the combinatorial mixer.
• Culture cells on chip successfully using rat cells as model system.

 

Future Directions

• Separating algae based on size. 
• Counting algae from sea water sample.
• Growing cells under combinatorial conditions using the combinatorial mixer
• Culture of Pseuo-nitzschia on the culture chip.
• Detection of toxin production off chip using ELISA.

People

Faculty:

• Prof. Yu-Chong Tai, EE and BE, Caltech
• Prof. Chihming Ho, MAE, UCLA
• Prof. Dave Caron, USC

Graduate Students:

• Mike Liu, Caltech
• Beth Stauffer, USC