Postdoctoral Researcher, Monash University
Babesia, a parasite similar to the malaria parasite Plasmodium, is the second most common bloodstream parasite found in mammals. It is transmitted to its vertebrate host by tick bites. Babesia bovis infects cattle herds in tropical regions worldwide and leads to mortalities, abortions and a reduction in meat and milk production. Tick control, prevention and treatment are expensive and only cost-intensive (polymerase chain reaction) or human error-prone (microscopy) diagnosis methods are available. Thus, a sensitive and reliable method for the diagnosis of babesiosis is highly desired. A number of optical spectroscopic techniques to detect the human malaria parasite were explored in our lab, including Attenuated Total Reflection Fourier transform infrared (ATR-FTIR) spectroscopy as a non-subjective diagnosic tool. Here, we demonstrate the applicability of spectroscopic methods for the detection of B. bovis. 1) First, we established ATR-FTIR spectroscopy as a method for the detection of B. bovis. As ATR-FTIR on blood samples is usually challenged by the background absorbance of blood components, blood samples were lysed thereby removing blood components. This increased the detection sensitivity from 77.3% to 92.0% for 0.25% parasitemia. 2) Using atomic force microscopy (AFM) IR, which allows for the collection IR spectra with a nanoscale spatial resolution, we could assign spectral features that are characteristic for parasites, i.e. DNA and lipid IR-bands, for example. 3) Synchrotron-based FPA-IR allows for IR imaging with the high intensity of synchrotron radiation. Averaging over the pixels in an image of B. bovis infected cattle red blood cells (cRBCs) and uninfected cRBCs yielded spectra corresponding to a whole infected or uninfected cRBC, respectively. The spectral changes from ATR-FTIR that account for the presence of B. bovis in cRBCs were confirmed by the use of AFM-IR directly on the parasite inside of a cRBC and by synchrotron-based FPA-IR on B. bovis infected and uninfected cRBCs. This proves that the diagnosis of B. bovis using ATR-FTIR spectroscopy is based on real changes in the biological composition of infected RBCs. Thus we show here the proof of principle of using IR spectroscopic techniques for diagnosing the threatening disease babesiosis.
[1.] L. Schnittger et al. Infec Genet and Evol. 12, 1788–1809 (2012) [2.] R. Bock et al. Parasitology, 129, S247–S269 (2004) [3.] Khoshmanesh, A. et al. Anal Chem, 86, 4379 (2014)
Abstract: New diagnostic modalities for malaria must have high sensitivity and be affordable to the developing world. We report on a method to rapidly detect and quantify different stages of malaria parasites, including ring and gametocyte forms, using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FT-IR) and partial least-squares regression (PLS). The absolute detection limit was found to be 0.00001% parasitemia (<1 parasite/μL of blood; p < 0.008) for cultured early ring stage parasites in a suspension of normal erythrocytes. Future development of universal and robust calibration models can significantly improve malaria diagnoses, leading to earlier detection and treatment of this devastating disease.
Pub.: 04 Apr '14, Pinned: 29 Aug '17
Abstract: The mechanical properties of agarose-derived hydrogels depend on the scaffolding of the polysaccharide network. To identify and quantify such higher order structure, we applied Raman optical activity (ROA)-a spectroscopic technique that is highly sensitive toward carbohydrates-on native agarose and chemically modified agarose in the gel phase for the first time. By spectral global fitting, we isolated features that change as a function of backbone carboxylation (28, 40, 50, 60, 80, and 93 %) from other features that remain unchanged. We assigned these spectral features by comparison to ROA spectra calculated for different oligomer models. We found a 60:40 ratio of double- and single-stranded α-helix in the highly rigid hydrogel of native agarose, while the considerably softer hydrogels made from carboxylated agarose use a scaffold of unpaired β-strands.
Pub.: 24 Mar '17, Pinned: 29 Aug '17
Abstract: Vibrational circular dichroism (VCD) spectra of aqueous solutions of proline were recorded in the course of titrations from basic to acidic pH using a spectrometer equipped with a quantum cascade laser (QCL) as an infrared light source in the spectral range from 1320 to 1220 cm(-1). The pH-dependent spectra were analyzed by singular value decomposition and global fitting of a two-pK Henderson-Hasselbalch model. The analysis delivered relative fractions of the three different protonation species. Their agreement with the relative fractions obtained from performing the same analysis on pH-dependent Fourier transform infrared (FT-IR) and QCL-IR spectra validates the quantitative results from QCL-VCD. Global fitting of the pH-dependent VCD spectra of L-proline allowed for extraction of pure spectra corresponding to anionic, zwitterionic, and cationic L-proline. From a static experiment, only pure spectra of the zwitterion would be accessible in a straightforward way. A comparison to VCD spectra calculated for all three species led to assignment of vibrational modes that are characteristic for the respective protonation states. The study demonstrates the applicability of QCL-VCD both for quantitative evaluation and for qualitative interpretation of dynamic processes in aqueous solutions.
Pub.: 25 Mar '14, Pinned: 29 Aug '17
Abstract: Changes in vibrational circular dichroism (VCD) were recorded on-line during a chemical reaction. The chiral complex nickel-(-)-sparteine chloride was hydrolyzed to free (-)-sparteine base in a biphasic system of sodium hydroxide solution and chloroform (CHCl(3)). Infrared (IR) and VCD spectra were iteratively recorded after pumping a sample from the CHCl(3) phase through a lab-built VCD spectrometer equipped with a tunable mid-IR quantum cascade laser light source, which allows for VCD measurements even in the presence of strongly absorbing backgrounds. Time-dependent VCD spectra were analyzed by singular value decomposition and global exponential fitting. Spectral features corresponding to the complex and free (-)-sparteine could be clearly identified in the fitted amplitude spectrum, which was associated with an exponential decay with an apparent time constant of 127 min (t(½) = 88 min).
Pub.: 14 Mar '14, Pinned: 29 Aug '17