Figure 1. Diagram of SPCE
The project will be co-supervised by Dr. David Inglis (research fellow) and Prof. Ewa Goldys.
Microfluidics research involves fabricating micron sized structures to handle fluids and facilitate chemical or biological tasks. Microfluidic or lab-on-a-chip devices are being used in a number of cutting edge biological industries such as DNA sequencing and medical diagnostics. Research in the field is multidiciplinary with most work focussed on making laboratory procedures cheaper and faster.
We are in a position to use recently developed techniques in microfluidics to sort and concentrate microbes. This will allow for earlier detection of dangerous cells in fluids ranging from drinking water to blood. Research using this technique has been used to separate different types of blood cells, including abnormally sized tumor cells, but has not yet been applied to micro organisms.
In this project the student will use the Macquarie University clean room to fabricate microbial and cell sorting devices as well as measurement and testing of the devices with non-infectious microbes. The student will develop skill in microfabrication, advanced microscopy and biology. These skills will be valuable in the growing field of biotechnology.
The microfluidics Phd project on offer is a full-time Macquarie University Research Excellence Scholarships (MQRES) with a current stipend of $20,007 p.a. tax exempt. Tenure is 3.5 years full-time, subject to satisfactory progress. Tuition fees will be sponsored for the scholarship tenure.
MQRES scholarships are available to domestic and international students. Prior to commencing, applicants should have completed a 4 year undergraduate degree in Engineering, Physics, Chemistry or Biology. Early career professionals interested in returning to higher degree studies are also encouraged to apply. Further information regarding this project, may be obtained by contacting the co-supervisor: Dr David Inglis, telephone: +61-2-9850-4167, email: dinglis@mq.edu.au. Application forms (see below) and conditions for awards are available from the Higher Degree Research Office, telephone +61-2-9850 7987, email: hdrschol@vc.mq.edu.au.
The new Nanobiophotonics PhD project on offer in 2008 will be carried out in partnership with the Department of Molecular Biology and Immunology, Health Science Centre, University of North Texas, Fort Worth, TX, USA.
1. Summary: The project will focus on detecting, enumerating and identifying low abundance proteins in physiological fluids which is a major challenge for medical diagnostics. This project will deepen the understanding of interaction of light with metal nanostructures, and, on this basis, a generic optical technology will be established for the detection of trace proteins in complex and dense physiological fluids such as whole blood. The highly sensitive technique developed in this project will be applicable to biomarkers at clinically relevant levels and it will remove the limitations of long analysis times, high instrumentation costs and the difficulty of real-time monitoring common in conventional methods.
The new method will be able to detect specific disease markers or to identify subtle differences in protein content in complex dense analytes such as blood and other body fluids, of practical utility in diagnostic and clinical situations. It will also be relevant in other areas such as medical diagnostics of viral diseases, and for unsolved environmental monitoring problems such as the presence of specific microorganisms in industrial waste. Owing to their design simplicity and low cost of components, the devices developed in this program will lend themselves well to the development of new commercial technologies for Australia.
2.1. Background - Surface plasmon resonance (SPR) SPR is an optoelectronic phenomenon that occurs when light internally reflects from an optical interface coated with a thin metal film at a specific angle [1]. Light incident to the surface interacts with electrons in metal forming collective excitations called surface plasmons that are sensitive to the refractive index (RI) in the adjacent medium within a narrow (~200 nm) region near the interface. This RI is related to the presence and mass of molecules in this region and it may be monitored by tracking the reflected light and its angular dependence.
The SPR technique plays a prominent role in the detection and analysis of surface-assembled thin molecular biolayers and, during the last decade, such biosensors have become extraordinarily successful [2]. For example, the SPR effect enables precise monitoring of binding occurring at biospecific surfaces on metal layers with extremely high sensitivity, better than 10-6. The review paper [3] summarises major developments in the SPR technology and its applications in chemical sensors [4,5]. Multichannel sensing has also been attempted [6], where the detection by SPR was accompanied by measurements of another effect such as fluorescence, absorption or Raman scattering. Important advances have recently been made in SPR detection owing to the discovery of nanostructure enhancement. It is possible, by using nanoparticles under carefully selected conditions, to considerably enhance the sensitivity of a conventional SPR device [7-9]. The present project aims to exploit this discovery for ultrasensitive whole blood immunoassays.
Figure 1. Diagram of SPCE
2.2. Background - Surface plasmon coupled emission (SPCE) This innovative fluorescence effect was explored in a series of recent pioneering works of the PIs [10-14]. SPCE can be observed using a transparent substrate such as glass coated with an ultrathin layer metal film (for example silver) that is coated with excited fluorophores distributed over the metal surface. Under such conditions, for fluorophores near the metal surface, a highly directional and polarised fluorescence emission is observed along the surface of a hollow cone, rather than isotropic as in conventional fluorescence (Fig 1). Moreover, the excitation is enhanced, due to the amplified evanescent field with enhancement factors of up to 100 easily achievable. Surprisingly, the effect has intrinsic spectral resolution. Important advantages of SPCE include improved excitation, light collection efficiency, discrimination of fluorescent background and high spatial selectivity in favour of surface-bound analytes.
2.3. Background- Immunoassays for protein detection Immunoassays are commonly used in medical diagnostics for highly specific detection of targeted proteins [15]. In this method an antibody is bound to a substrate such as a permeable membrane, surface of a well plate or an SPR chip. In the presence of antigens the bioaffinity reaction is reported either by change of color, fluorescence or reflectivity. The two most common immunoassay formats, membrane and well plate (ELISA) assays, despite widespread use, suffer from drawbacks particularly apparent in the analysis of complex biological fluids such as blood. These disadvantages effectively prevent rapid sensing of low abundance analytes in whole blood including important bio- or disease markers. For example, in membrane bioassays blood needs to be separated prior to analysis and, during this process, biomarkers of interest may bind to the separated components that are not analysed, or they may adhere to the membrane instead of reaching the analysis spot, all of which produce incorrect readings. In the ELISA method, whole blood is placed in the well where blood components react with an antibody located at the well surface. As blood is optically dense, the readout requires its time-consuming removal after conjugation [16-20]. The SPR sensor, despite the use of a blocking protein, suffers from non-specific binding, especially at high sensitivity limits. In the assay format proposed here none of these issues will be relevant as whole blood will be analysed without the separation step nor washing, and non-specifically bound proteins will not contribute to the observed signal from the fluorescently labelled analyte.
3. Outcomes At the conclusion of the project we plan to achieve the following outcomes:
Application of the optimised nanostructures for whole blood bioassays for important biomarkers, at sensitivity levels between 0.1 and 1 ng/ml.
4. Skills to be developed by the student The PhD graduate will become familiar with optics, optoelectronics and fluorescence technologies. He/she will gain good practical exposure to nanotechnology. With the aid of advanced commercial software RSOFT they will further develop their skills in mathematical modelling of SPR/SPCE effects. This mathematical modelling will be an integral part of the project. He/she will become familiar with bioassay technology. An extended period of study in the US will also form part of this program.
Research Project SPRSPCEReferences
[2] Liedberg, B. et al. (1995), Biosens. & Bioelectr. 10:i-ix; Sambles, J. R. et al. (1991), Contemp. Phys. 32:173-183.
[3] Homola J. Yee SS. Gauglitz G. Sensors & Actuators B-Chemical, vol.B54, no.1-2, 25 Jan. 1999, pp.3-15.
[4] Wilson DM. Hoyt S. Janata J. Booksh K. Obando L.IEEE Sensors Journal, vol.1, no.4, Dec. 2001, pp.256-74.
[5] Melendez, J. (1997), Sensors and Actuators B 39:375-379).
[6] Nenninger et al. (1998),Sensors and Actuators B 51:38-45
[7] "Nanoparticles .." San Jose, CA, USA. SPIE. U.S. Air Force Office of Sci. Res. 24-25 Jan. 2001 , SPIE-Int. Soc. Opt. Eng. Proceedings of SPIE, vol.4258, 2001, USA.
[8] Chah S, Hutter E. Roy D. Fendler JH. Yi J. Chemical Physics, vol.272, no.1, 1 Oct. 2001, pp.127-36.
[9] Hongxing Xu. Kall M. Sensors & Actuators B-Chemical, vol.B87, no.2, 10 Dec. 2002, pp.244-9.
[10] Matveeva, E, Gryczynski, Z, Gryczynski, I. and Lakowicz, J.R. (2004). J. Immunol. Methods, 286, 133-140.
[11] Matveeva, E., Malicka, J., Gryczynski, I., Gryczynski, Z., and Lakowicz, J.R. (2004). BBRC, 313, 731-736.
[12] Malicka J., Gryczynski I., Gryczynski Z., Lakowicz J.R. (2004). J. Biomolecular Screening, 9(3), 208-2016.
[13] Calander, N.. Annal. Chem. 2004, 76, 2168-2173
[14] Gryczynski I., Malicka J., Gryczynski Z., Nowaczyk K., Lakowicz J.R (2004). Anal. Chem., 76, 4076-4081.
[15] Vo-Dinh T, Sepaniak MJ, Griffin GD, Alarie JP. Imunomethods 1993, 3:85-92
[16] von Lode P, Rosenberg J, Pettersson K, Takalo H.. Anal Chem. 2003, 75(13):3193-3201.
[17] Choi S, Choi EY, Kim DJ, Kim JH, Kim TS, Oh SW. Clin. Chim. Acta 2004, 339(1-2):147-156.
[18] von Lode P, Rainaho J, Pettersson K. Clin. Chem. 2004, 50:1026-1035.
[19] Ahn JS, et al. Clin. Chim. Acta 2003, 332(1-2):51-59.
This new PhD project will focus on developing automated methods of microbial identification based on data mining of laser scanning microscopy images.
Higher organisms such as animals and plants can be easily recognised by their appearance but many unicellular organisms such as yeasts are very difficult to identify in this way, especially when they are closely related as in the case of strains of a particular genus. Part of the challenge of specifying the standard cell appearance for a single yeast strain is dealing with heterogeneous populations, where features of individual cells such as size and shape can vary. The existing procedures for the distinction of yeast strains, including Saccharomyces cerevisiae (baker's and brewer's yeast) depend either on metabolic or genetic markers, or on the properties of colonies. Up to now, rapid methods of species recognition using only a small number of cells and applicable to mixed strain populations were not available, despite advances in the underpinning techniques. Earlier we were able to produce, for the first time, quantitative characteristics of selected strains of Saccharomyces cerevisiae by exploiting the wealth of information contained in fluorescence microscopy images of unstained cell populations and using new software technologies. We used image processing tools which were specially developed by modifying a freeware program ImageJ.
The project will focus on using the methodology developed earlier in our group and focus on the yet untouched characteristics of yeast, such as NADH fluorescence which is related to yeast metabolism and can therefore help distinguish live form dead cells as a well as look for any multiphoton signatures of cells which have not been examined yet. We also plan to use the technology to assess the conditions of microcolonies, whose images will be supplied by the Westmead Hospital.
Skills to be developed by the PhD student will include in-depth familiarity with advanced laser scanning microscopy within the Optical Characterisation Facitity at Macquarie University. After a period of study he/she will need to be able to write additions to freeware software and use statistical software packages. Readiness to read and understand some mathematics and statistics will be necessary as well. He/she may need to gain familiarity with yeast cultivation protocols, and will need to read some biotechnology publications to understand microbial physiology. As this is a cross-disciplinary program no one is really well prepared for all this, but a bachelor degree in Electrical engineering or Physics may be very suitable, as well as other Science and even applied mathematics degrees, depending on individual interests and courage and willingness to learn new things. The microscopes are software driven and easy to run and the yeast is happy to grow for anyone.