Lead Investigator

Chih Ming Ho, UCLA

Overview

The laser-induced fluorescence detection system is going to be used to detect domoic acid (DA) downstream of the algae culture chip which is developed in Caltech. Assay prepration is done by Dr David Caron’s group in USC. Traditional competitive ELISA assay cannot be used with the chip because of not high enough sensitivity. In ELISA concentrations lower than 0.2 ng/mL cannot be detected and the concentration needed on the chip is 0.08 ng/mL or nM.

Approach

The ultra sensitive optical sensor based on laser induced fluorescent combined with confocal microscopy will provides a very small detection volume with which concentrations down to fM can be detected. In this method, molecules of interest are labeled with fluorophores and by flowing the sample in the chip they will pass the detection volume of the sensor. A photon burst is observed when the molecule passes the detection volume and the number of peaks per unit of time indicates the concentration of sample under study. Using the pinhole for confocal microscopy will reduce the background noise coming from the reflection and spurious fluorescence background and going to lower concentration down to single molecules will be made possible. With this method different kinds of molecules can be detected as long as they are differentiated from the solution by tagging them with fluorophores. The detection part being separate from the chip makes it very easy to replace the chip or the sample to be used with this sensor.

Systems/Experiments

Figure 1: Schematic of the system

The setup consists of an Ar Ion laser for excitation. The laser light is reflected on the dichroic mirror and goes through the objective shining on the sample and exciting the fluorophores. The same objective collects the light from the sample. Figure 1 is a schematic of the system.

Since its wavelength is different, the emitted light goes through the dichroic mirror towards the detection system. The detection part consists of a spatial filter, which consists of two lenses and a pinhole. The role of the pinhole is to make the setup confocal. Finally, another lens focuses the light on the Avalanche Photo Detector (APD). The objective used is a high numerical aperture 100X, NA=1.3 oil immersion objective.  The reason for this objective is that a small sample volume is needed both for single molecule detection and also for noise reduction from the spurious fluorescence from the Raman scattering of the solvent molecules.

The signal from the emission is passed through a spatial filter which consists of a focusing lens a pinhole and another lens. The final lens will focus the signal on the detector (APD).

Future Directions

The tests are going to be done in a microchannel versus a droplet. We will start working with the real sample.

People

Faculty:

• Dr Chih-Ming Ho, Mechanical and Aerospace Engineering, UCLA

Graduate Students:

• Leyla Sabet, Mechanical and Aerospace Engineering, UCLA