Novel Assays and Portable Instrument for Label-free Toxin Detection
Moran Bercovici, Govind V. Kaigala, Robert D. Chambers,
Supreet S. Bahga, Christopher J. Backhouse and Juan G. Santiago
Current methods for environmental monitoring such as gas and liquid chromatography coupled with mass spectrometry are considered sufficiently sensitive and accurate. However, their use is mostly confined to laboratory settings because of their size, required amount of sample preparation, power consumption, etc. There is a need for detection techniques that are cost-effective, sensitive, and portable. To this end, we have developed novel fluorescence based indirect detection techniques and demonstrated their use for toxin detection in a portable hand-held device.
Low Cost Hand-held device for Electrophoresis and Isotachophoresis
We have developed an inexpensive hand-held device (240 g) that implements microchip electrophoresis and isotachophoresis (ITP) with laser induced fluorescence (LIF) detection. This self-contained instrument, shown in Figure 1, integrates the functionality required for high voltage generation onto a microelectronic chip, includes LIF detection and is powered by a universal serial bus (USB) link connected to a laptop computer. We have shown that the device can provide quantitative analysis of fluorescent species, with a limit of detection of 100 pM. We have also integrated several ITP assays in the device for label-free detection of wide variety of chemicals, including chemical warfare agents, explosives and endocrine disruptors, with no sample processing.
Figure 1: The hand-held ITP instrument (dimensions: 7.6 ×5.7× 3.8 cm) is powered using a standard USB link connected to a laptop computer. The device is self-contained and includes a 5 mW laser, a photodiode, high voltage generation, switching, and communication functionality. The metal casing acts as a Faraday cage to reduce the effects of environment noise.
Fluorescent Nonfocusing Tracer (NFT) Assay
We have developed a novel fluorescence based assay for visualizing various isotachophoresis (ITP) zones. We introduce negligibly small concentrations of a fluorophore that is not focused by isotachophoresis. This nonfocusing tracer (NFT) migrates through multiple isotachophoresis zones. As it enters each zone, the NFT concentration adapts to the local electric field in each zone. ITP zones can then be visualized with a point detector or camera. The method can be used to detect, identify, and quantify unknown analyte zones and can visualize complex and even transient electrophoresis processes. We also have demonstrated separation and detection of analytes using this assay in our hand-help ITP device (see Figure 2).
Figure 2: Demonstration of separation and NFT-based indirect detection of three model analytes using the hand-held device. The right-hand column presents simulation results (using our Spresso simulation tool) and the left-hand column present experimental results. Both plots show detectable zones of bistris (BT), tris (TR) and histidine (HST) focused between LE (sodium-Bes)and TE (pyridine-Bes). Mixing of an additional species with TE results in a new ¿step¿ in the fluorescence signal.
Fluorescent Carrier Ampholytes (FCA) Assay
We have developed a novel indirect-detection technique that allows the detection of analytes with little a priori knowledge of their electrophoretic mobilities. The technique is based on the displacement of fluorescently labeled carrier ampholytes by focused analytes in ITP. The analytes are detected indirectly and quantified by analyzing the gaps in the fluorescent ampholyte signal. We have integrated this assay with our hand-held ITP device and demonstrated detection of 2 nitrophenol (2NP), and 2,4,6-trichlorophenol (TCP) in tap water (see Figure 3), without labeling or sample preparation steps. We have also applied the FCA assay with hand-held device to detect indirectly (label-free) ionic water soluble explosives, ammonium 2,4,6-trinitrophenolate (Dunnite) and 2,4,6-trinitrophenol (TNP), and a herbicide, dichlorophenoxyacetic acid (2,4-D) in river water.
Figure 3: Detection of unlabeled 2 nitrophenol (2NP), and 2,4,6-trichlorophenol (TCP) in tap water using the handheld ITP device. (a) The control case shows the distribution of fluorescent CAs in the tap water; (b) 50 µM of 2NP is mixed with the TE and creates a zone between the LE and TE (the zone displaces labeled CAs, resulting in a new gap in the signal). (c)10 µM of TCP is mixed with TE and creates a gap at a different location. (d) Both 2NP and TCP are added to TE and create two distinct gaps in the fluorescence signal.
As described by Kaigala et al. , our current limit of detection for this device is order 10 µM for unlabeled analytes (100 pM for fluorescence species). We are currently working on methods to improve the sensitivity of these assays including variations of cross-sectional area of ITP channels , minimizing dispersion of ITP processes , and cascade ITP methods (as yet unpublished).
Summary of chemicals demonstrated with ITP-based indirect detection
We have demonstrated detection of a variety of unlabeled analytes with no sample preparation in drinking water or river water. The following are examples:
- 2 nitrophenol and 2,4,6 trichlorophoenol (US EPA priority pollutants) (in portable device) 
- 4 Dichlorophenoxyacetic acid (common herbicide, implicated endocrine disruptor) 
- Saxitoxin (a schedule 1 chemical warfare agent) (unpublished)
- Explosives: trinitrophenol, picric acid, picramic acid, and dunnite (unpublished)
- Kaigala G.V., Bercovici, M., Backhouse, C.J., Santiago, J.G., Lab Chip 2010, 10, 2242-2250.
- Bahga, S.S., Kaigala, G.V., Bercovici, M., Santiago, J.G., Electrophoresis 2011, 32, 563-572.
- Garcia-Schwarz, G.; Bercovici, M.; Marshal, L., Santiago, J.G., J. Fluid Mech. 2011, 679, 455-475.
- Bercovici, M., Kaigala G.V., Sanitago, J.G., Anal. Chem. 2010, Anal. Chem. 2010, 82, 1858-1866.