Chapter 6: Applications of Microfluidics in Marine Microbiology
6.1 Introduction
The uses of microfluidic devices are not limited to cell culture and cell-based assay for mammalian cells as discussed in previous chapters. Any other types of cells can take advantage of the benefits that microfluidic devices offer: high-throughput screening, portability, inexpensive platform, and reduced sample volume and reagents.
Under the collaboration with Dr. David Caron’s group at University of Southern California’s Department of Biological Sciences, one area that our group has applied microfluidic devices to is in the field of marine biology, specifically in studying algal bloom and toxin production by algae. Algal bloom and toxins produced by different algae have always caused problems to the environment and marine ecology. Due to certain favorable changes in environmental conditions, some algae can increase to a large density in a short period of time. For example, many studies have shown a strong correlation between phosphorus loading and phytoplankton production in freshwater [118]. Some produced toxin and can directly cause sickness or death of organisms such as shellfishes that feed on the algae. The phosphorus can originate from excess nutrients in fertilizers that enter the river or sea during water runoff. More importantly, when large quantity of those contaminated shellfishes was consumed by organisms in higher order of the food chain, fatalities often resulted. For example, Alexandrium tamarense, can produce saxitoxin and cause paralytic shellfish poisoning [119]. Non-toxic algal bloom can lead to harmful effects by disrupting food chains.
Pseudo-nitzschia is one type of algae that produces a neural toxin called domoic acid, which when transferred through the food chain causes sickness and mortality in marine mammals, seabirds and even humans [120]. Perl et al. reported an outbreak of domoic acid intoxication in Canada in late 1987 that led to illness of 107 adults and death of three elderly people [121]. Mussels were contaminated and resulted in amnesic shellfish poisoning with symptoms of gastrointestinal and neurological disorders. Also, domoic acid has been linked to the death of numerous sea lions along the California coastline in 1998 [122].
Domoic acid is a neural toxin that affects animals with complex central nervous systems. Domoic acid is structurally analogous to neural transmitter glutamic acid, which is present in nearly 40% of all neuronal synaptic sites, and upon binding to receptors, domoic acid prolongs receptor activation and excessive cation influx. This uncontrolled influx can cause nerve cells to degenerate. During Pseudo-nitzschia bloom, domoic acid is not always produced. In another word, growth of algae does not equal domoic acid production. Studies done by other groups have suggested that many factors might induce or suppress algae to produce toxin. Maldonado el al. reported that deficiency in iron and copper can induce domoic acid production [123]. Silicate depletion has been shown to cause increase in domoic acid production [124]. It has been suggested that high phosphate level can lead to increased domoic acid production [125].
Bacteria strains have also been shown to increase domoic acid production by as much as 95-fold [126]. Yet, exact causes are unclear and there are numerous possible factors that can cause toxin production. To completely elucidate the causes of toxin production,
many potential compounds will have to be screened. This leads to an enormous number of experiments to be performed and large quantity of reagents and cells to be used.
To facilitate progress in marine biology research and speed up the process of screening for possible factors inducing toxin production, we would like to make a chip to culture Pseudo-nitzschia under different growing conditions. The toxin will be detected using ELISA method or an ultra sensitive optical sensor being developed at Dr. Chih- Ming Ho’s lab at University of California at Los Angeles. The current state-of-the-art detection technology indicates that per cell toxin load may range over 2 or 3 orders of magnitude but its sensitivity is limited since a sample size of at least 100 cells/mL is required. The new optical sensor will be able to push the sensitivity to 10 cells/mL or to even single molecule of domoic acid. The algal cells will be trapped and cultured on- chip and combinatorial mixer discussed in chapter 3 and 4 will be used to expose algal cells to different conditions. Cellular contents will be extracted for detection of toxin from lysates.
In this chapter, the development of the applications of microfluidic cell-based assaying device for studying toxin production by algae is discussed. As most algal cells are not adherent like the mammalian cells used in previous experiments, on-chip traps were needed to contain floating cells like algal cells inside the micro chambers. Various trapping designs will be presented and the developed trapping protocol will be discussed.
The method to maintain algae in a healthy state and to culture algae on-chip has been developed. As the presence of the intracellular toxin, domoic acid, cannot be directly monitored on-chip, a method to lyse the cells on-chip is presented.