Overview

The marine application area during the first three years of CENS was divided into two projects, one of which was focused on the detection of brown tide algae (BTA). This project was selected for termination by the CENS leadership at the end of the third year, but was allowed to continue at much reduced funding for an additional semester, to enable students to finish their theses, to write up the results, and generally to wrap up the project. The results of the project not contained in the Year 3 report are summarized below.

Approach

The Atomic Force Microscope (AFM) is a highly sensitive instrument capable of detecting single molecules based on their affinity for a functionalized tip. Identification of single-cell microorganisms such as the brown tide alga (BTA) by measuring forces with an AFM is conceptually very simple. The microorganisms are attached to a flat surface, and the tip of the AFM is functionalized with monoclonal antibodies (MAbs) to the organisms. The tip is moved towards the cell surface until it touches it, and then retracted. A force-distance (f-d) curve is obtained by measuring the deflection of the cantilever that occurs as the tip moves towards and away from the surface. If there is an antibody-antigen (Ab-Ag) binding event, this bond must be broken for the tip to retract to its original position, and this can be found from the f-d curve. If a MAb-Ag bond is broken, the Ag must be present on the cell surface, and therefore the cell must be a brown tide alga. (Note that this approach works for arbitrary cells, provided that monoclonal antibodies to them are available.) To implement this detection scheme requires that we attach cells to a substrate surface, and that we functionalize an AFM probe tip with Abs. Tip fictionalization was adequately described in the Year 3 report.

Accomplishments

A major difficulty encountered in this project was our inability to attach the BTA to a substrate surface. While most cells (e.g. mammalian cells) attach readily to many surfaces, the BTA do not. We finally solved the problem by using a polycarbonate membrane with a pore size comparable to the dimensions of the BTA. Figure 1 shows a BTA cell partially inserted into a pore. This is done by reducing the pressure on one side of the membrane to “aspirate” the cells and confine them to the pores. This is a mechanical, non-specific technique that can be used for virtually any cell of the same approximate size.

Figure 1: AFM image of a BTA cell in a pore of a polycarbonate membrane.

With this immobilization technique we ran force-distance experiments with BTA and controls. As controls we used the bare surface and also two algae of sizes comparable to the BTA. The results are shown in the histograms of Figure 2. Bond rupture forces below ~ 100 pN are non-specific. The figure shows clearly that the BTA can be identified with high signal to noise ratio from the histograms. As argued in the Year 3 report, this technique has single-cell resolution and compares favorably in speed to other approaches which have coarser resolutions. In addition, it is potentially capable of in situ detection or even chip implementation by coupling it with microfluidics and MEMS technology and using piezoresistive cantilevers that do not require optical readout.

Figure 2: Histograms of rupture forces for several controls and for the BTA cells.

People

David Caron, Professor
Ari Requicha, Professor, USC
Mark Thompson, Professor
Chongwu Zhou, Professor
Mrinal Mahapatro, Dr.
Pamela Gross, Dr.
Beth Stauffer
Alex Lee, GSR