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Posted: Nov 29, 2011

A fast nanotechnology platform to detect/capture bacteria in clinical samples

(Nanowerk Spotlight) Surface-enhanced Raman spectroscopy (SERS) is a powerful research tool that is being used to detect and analyze chemicals as well as a non-invasive tool for imaging cells and detecting cancer. It also has been employed for label-free sensing of bacteria, exploiting its tremendous enhancement in the Raman signal.
SERS can provide the vibrational spectrum of the molecules on the cell wall of a single bacterium in a few seconds. Such a spectrum is like the fingerprints of the molecules and therefore could be exploited as a means to quickly identify bacteria without the need of a time-consuming bacteria culture process, which typically takes a few days to several weeks depending on the species of bacteria.
To practically apply SERS to the early diagnosis of bacteremia – the presence of bacteria in the blood – it is most desirable to be able to capture bacteria in a patient's blood onto the SERS substrate.
"A typical SERS-active substrate consists of arrays of nanoscale metallic objects, for example, silver nanoparticles and etch-pits on silver surfaces, which can sustain surface plasmon polariton resonance and enhance the Raman signal of molecules on or near the substrate," Yuh-Lin Wang, Distinguished Research Fellow, Institute of Atomic and Molecular Sciences, Academia Sinica, and Professor in the Department of Physics at National Taiwan University, explains to Nanowerk. "In our recent work, we found that coating a thin layer of vancomycin on a SERS substrate drastically increases its capability to capture bacteria in the blood samples without introducing significant spectral interference to SERS spectrum of the captured bacteria."
Previously, researchers already used vancomycin-coated magnetic nanoparticles to capture bacteria in water. Wang and his team therefore asked the question whether it is possible to endow the vancomycin-coated SERS substrates with the concurrent functionalities of bacterial capturing and sensing.
Reporting their work in the November 15, 2011 issue of Nature Communications ("Functionalized arrays of Raman-enhancing nanoparticles for capture and culture-free analysis of bacteria in human blood"), first-authored by Ting-Yu Liu, they demonstrate that functionalization by vancomycin of substrates of silver nanoparticles on arrays of anodic aluminum oxide nanochannels not only dramatically enhances their ability to capture bacteria in liquid but also significantly increases their SERS signal.
Capture and analyze bacteria in blood by a dual function biochip
Capture and analyze bacteria in blood by a dual function biochip. A team led by Yuh-Lin Wang has demonstrated a dual function biochip that can not only effectively capture bacteria in human blood but also enhances the Raman signals of the bacteria, allowing their analysis by surface enhanced Raman spectroscopy (SERS). The core of the biochip is an array of silver nanoparticles (silver) coated by Vancomycin (green), on which bacteria (white) are selectively captured while blood cells (red) are excluded. (Image: Dr. Wang, Academia Sinica. Correspondence and requests for materials should be addressed to Prof. Wang, email: ylwang@pub.iams.sinica.edu.tw)
Wang notes that the team's findings took them by surprise since the signals from the vancomycin coating were expected to be larger than that from the bacteria cell wall since the later is closer to the SERS substrate.
"This unexpected discovery opens up many possibilities for the creation of SERS-based multifunctional biochips for rapid culture-free and label-free detection and drug-resistant testing of microorganisms in clinical samples," he says.
Coating SERS substrates by vancomycin, which is an antibiotic by itself, in order to add the function of capturing bacteria to the substrates is a brand-new approach to overcome a major obstacle facing the practical application of SERS in clinical diagnosis.
"We have been making various attempts to overcome this obstacle in the last two years" says Wang. "This exciting result is the outcome of an experiment that makes some sense superficially but is considered unlikely to work on second thought because of the likely interference from the vancomycin coating. After we saw the surprisingly good result that the SERS signals from the vancomycin coating do not interfere with that of the captured bacteria, we pondered on the question "why" for months and finally came up with some qualitative explanation as given in our paper."
"In retrospect, this is an interesting case in which the original motivation was to tackle the conceptually simple and practically challenging problem of trying to capture bacteria onto a small area of a biosensor without compromising its original sensing function. It turns out that our solution not only allows us to capture bacteria ~1000 times more effectively but also enhances the sensitivity of the sensor by several times rather than decreasing it."
To demonstrate the bacterium-capturing capability of their substrate, the researchers immersed it in a water sample with ultra-low concentration (102 cfu ml-1) of bacteria for 1 hour and then rinsed it in deionized water. Examining the substrate with scanning electron microscopy (SEM), they were then able to detect a concentration of bacteria on their substrate.
Wang and his team point out that their findings are a major step towards the development of a high-speed and -sensitivity nanotechnology platform that has high potential to capture/detect bacteria in clinical or environmental samples.
This SERS-based rapid detection method is a very promising approach to dramatically reducing the time needed to detect bacteria in the blood of bacteremia patients to within an hour. By contrast, a conventional biological assay usually requires the sample preparation time ranging from days for fast growing bacteria to weeks for slow growers.
The researchers point out that, In principle, this sensing platform could be exploited for the detection of various microorganisms such as virus and bacteria in various clinical samples, e.g., water, phlegm, sputum, blood and marrow, as well as food, and environmental samples.
"Culture-free and label-free detection of microorganisms remain among the most exciting directions in the development of rapid biosensing technology," says Wang. "One of the most difficult challenges is the development of complementary sample preparation technologies. In order for a new method to be accepted by the research community, scientists and practitioners are looking for a total rather than partial solution."
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