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The potential for a rapid, field-deployable, automated biosensor detection system for pathogens


Dr Rosalie Multari (a) and Dr John Waite (b)


Hospital acquired infections (HAI), often referred to as ‘superbugs’, are never far from the news. MRSA (methicillin-resistant Staphylococcus aureus) leads the way in press coverage but is just one of a growing group of bacteria that can cause diseases with increasingly limited therapeutic options. Organisms with resistant strains include vancomycin resistant Staphylococcus haemolyticus, Clostridium difficile (C. diff), and numerous enterococcal isolates with plasmid-mediated gentamicin resistance and vancomycin resistance.


At a time of rising levels of MRSA and other hospital-acquired infections, rapid and reliable detection would offer an important safeguard to patients and staff alike.


Laser-Induced Breakdown Spectroscopy


Laser-induced breakdown spectroscopy (LIBS) is a spectroscopic analysis technique in which a focused laser pulse is directed onto a target. Whether a solid, liquid, or gas, the energy from the pulse vaporises, atomises and ionises the target material to form a micro-plasma, which emits light as a result of relaxation of electrons from excited to lower energy states. The spectral signature of the plasma holds the characteristic optical fingerprint of the individual elements within the target (Figure 1).


LIBS and Biological Samples


The LIBS group at Applied Research Associates is investigating the use of LIBS in a number of novel testing situations, including industrial process monitoring, environmental monitoring, and workplace surveillance for harmful materials, as well as deployment in space exploration.


In 2006, Matthieu Baudelet and colleagues [1] used processed LIBS spectra captured through a Mechelle Echelle spectrograph (Andor) to investigate the relative concentration of six trace elements in pure samples of five bacterial species and showed the equipment’s suitability for accurate identification and discrimination.


Figure 1. LIBS plasma plume generation


Figure 2. The ARA team: (left to right): Rosalie A. Multari, David A. Cremers, and Melissa L. Bostian


The laser pulse usually lasts for 5 to 20 ns and, typically, the emitted light is routed to an Echelle spectrograph, with its combination of high resolution and wide wavelength coverage. The spectrum observed in the first 100 ns is dominated by continuous, intense, white-light radiation that contributes to a high background signal, making the observation of discrete lines difficult. The plasma plume expands with time and the excited species relax further.


After a delay of approximately 1 µs from the incident laser pulse, discrete spectral lines originating from various ionic species become increasingly visible as the high


background emissions decay. The exact timing and the spectral lines vary with the type of sample, the distance from the centre of the plasma and the wavelength of the incident laser light. Typically, the evolution of the plasma and the changes in its content occur on a microsecond timescale and a precision, ultrafast camera is required – usually an intensified CCD (ICCD) system.


If the laser selected exhibits a good Gaussian profile, which allows focusing to a near diffraction-limited spot, LIBS does not require a large amount of energy. Typically, energies of only a few tens of mJ are necessary. In many ways, the analysis is similar to that performed by an ICP (Inductively Coupled Plasma) analyser but without the sample preparation that ICP requires.


It is the combination of minimal sample preparation and a low power requirement that makes LIBS a potentially valuable technique for automated use in the field and or unattended situations outside the controlled laboratory environment.


The ARA group (Figure 2) used the iStar intensified CCD camera (Andor) and Echelle spectrograph to demonstrate for the first time that LIBS may be used to discriminate pure, viable pathogen samples based only on raw (unprocessed) LIBS spectra. This work was carried out in a collaborative research effort with a group led by Dr. John Gustafson of the New Mexico State University Biology Department (Figure 3). This collaborative work is the first blind study in which LIBS data was used to successfully identify five pathogenic bacterial samples and differentiate between strains of a multiple-antibiotic-resistant species [2]. The LIBS apparatus used to collect the data as set-up in the ARA laboratory is shown below (Figure 4).


Sub 60-second identification of MRSA and other HAIs


Multiple-antimicrobial-resistant methicillin-resistant Staphylococcus aureus (MRSA) causes serious infections in hospital patients and within the general populace and strains exhibiting reduced susceptibility to the principal anti-staphylococcal drug, Vancomycin, have been reported. Numerous time-consuming culture-media-based and molecular biology techniques are usually required to differentiate common bacterial pathogens, resolve their clonal relationship among single specie strain collections, and determine antimicrobial resistance profiles. Determining antimicrobial resistance phenotype is imperative when determining which antimicrobial regimen will best suit a diseased individual.


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