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Bringing safe water to the masses
A fresh approach using Raman spectroscopy enhanced with nanoparticles may provide a sensitive technique for detecting individual cells of water-borne pathogens such as Cryptosporidium.
by Peter J VIKESLAND and Krista L RULE

uman population growth and the changes in global water cycles owing to global warming severely stress the planet’s freshwater supply. With this pressure comes an increased risk of contamination of water sources used for drinking. Accordingly, researchers have recently put much effort into the development of improved tools and techniques to detect and quantify chemical and biological drinking water contaminants. An ideal technique would be deployable in the field and provide the necessary sensitivity and specificity to protect human health.

One promising avenue of investigation comes from nanotechnology-enabled sensor platforms. A team of researchers at Virginia Polytechnic Institute and State University (Virginia Tech) in the US employs a nanoparticle-based immunoassay for the capture and detection of pathogens in drinking water. Immunoassays are tests that measure the concentration of a substance by looking at the interaction between an antigen (that is, the pathogen) and a complementary antibody. Although immunoassays have found a wide range of applications in clinical biology — such as diagnostic tests for HIV and West Nile Virus — they have been less frequently used in drinking-water systems.

In the Virginia Tech protocol, the Department of Civil and Environmental Engineering researchers immobilise antibodies specific to pathogens commonly found in drinking water on the surface of a membrane filter (see figure on page 29). As a water sample passes through the antibody-functionalised filter, the immobilised antibodies capture the pathogens. Following filtration, researchers wash the membrane-filter surface to rid the assay of extraneous debris. The surface of the membrane then undergoes exposure to an antibody–gold conjugate (or immunogold), which consists of gold nanoparticles coated with antibodies specific to the pathogen of interest as well as fluorescent-dye molecules to label the pathogen. When the antibodies on the immunogold labels bind to the captured pathogens, a so-called immuno-sandwich forms. Washing gets rid of unbound immunogold particles, leaving only the labels attached to the pathogens.

The investigators employ Surface Enhanced Raman Spectroscopy (SERS) to detect the pathogens trapped on the filter. Raman Spectroscopy alone does not provide a sufficiently sensitive detection technique. However, when Raman-active molecules, such as the fluorescent dyes, are in the vicinity of noble metal nanoparticles or a roughened noble metal electrode, a significant increase in the intensity of the Raman signal occurs. This effect, known as surface enhancement, can lead to signals as much as 1014 times the intensity of the normal Raman signal. This signal enhancement can, under some conditions, enable single-molecule detection.

Quantification of sample fluorescence remains a common strategy in ultrasensitive immunoassay methods. However, one benefit of using SERS detection lies in the fact that Raman absorption bands are typically much narrower relative to those used in fluorescence spectroscopy. These narrower bands favour multi-analyte detection because of less overlap of different spectra. Another advantage of SERS is that signals are less subject to photobleaching, an effect that often leads to decreases in fluorescence signals within the time required to take a single measurement.

The team conducts initial tests of this sensor platform on the protozoan parasite Cryptosporidium parvum. Transmission of Cryptosporidium poses a significant health risk in both developed and developing regions of the world. This organism, present in most natural waters, afflicts both humans and animals indiscriminately. Infectious doses fall in the range of 10–1,000 oocysts (the encysted form the organisms take to transfer them to new hosts) for healthy persons and as low as 10–100 oocysts for immuno-compromised individuals. Cryptosporidium oocysts strongly resist disinfection by free chlorine or other common disinfectants. Drinking-water treatment plants therefore resort to chemical pretreatment and filtration to effectively remove them from treated water. Given these issues, Cryptosporidium represents an ideal candidate to test the usefulness of a SERS-based detection strategy.

The potential automation of the technique when coupled with molecule-specific absorption spectra may enable the SERS immunofilter method to be used for simultaneous detection of several pathogens. Existing protocols for drinking water pathogen detection typically require extensive processing and sample preparation. In contrast, the immunofilter technique only requires sample filtration, rinsing of debris, and addition of the immunogold, yet achieves similar detection levels comparable to existing methods. Futhermore, the possibility exists to incorporate numerous commercially available fluorescent dyes, each with its own Raman-spectrum fingerprint, into an immunogold label. Immunogold conjugates can therefore be synthesised with different dyes and antibodies specific to each type of pathogen of interest.

In a multi-analyte test, the user would expose the surface of the immunofilter to several types of immunogold and then measure it with a Raman spectrometer. The observed fingerprints could then indicate the pathogens present in the water samples.

As Raman spectrometers become more affordable — some field instruments cost from US$10,000–$20,000 — small analytical laboratories and municipalities could potentially afford Raman spectrometers. They could adopt this new immunofilter method to monitor for pathogens in finished drinking water on site instead of sending bulky grab samples to expensive analytical laboratories and then waiting for up to two weeks for results. A simple, rapid method for pathogen detection adapted from this fresh strategy would lead to safer drinking water. Currently optimising the system within the laboratory, the researchers aim to move on to field trials once they complete the studies.

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