A nanoporous interferometric micro-sensor for biomedical detection of volatile sulphur compounds
© Kumeria et al; licensee Springer. 2011
Received: 14 September 2011
Accepted: 16 December 2011
Published: 16 December 2011
This work presents the use of nanoporous anodic aluminium oxide [AAO] for reflective interferometric sensing of volatile sulphur compounds and hydrogen sulphide [H2S] gas. Detection is based on changes of the interference signal from AAO porous layer as a result of specific adsorption of gas molecules with sulphur functional groups on a gold-coated surface. A nanoporous AAO sensing platform with optimised pore diameters (30 nm) and length (4 µm) was fabricated using a two-step anodization process in 0.3 M oxalic, followed by coating with a thin gold film (8 nm). The AAO is assembled in a specially designed microfluidic chip supported with a miniature fibre optic system that is able to measure changes of reflective interference signal (Fabry-Perrot fringes). When the sensor is exposed to a small concentration of H2S gas, the interference signal showed a concentration-dependent wavelength shifting of the Fabry-Perot interference fringe spectrum, as a result of the adsorption of H2S molecules on the Au surface and changes in the refractive index of the AAO. A practical biomedical application of reflectometric interference spectroscopy [RIfS] Au-AAO sensor for malodour measurement was successfully shown. The RIfS method based on a nanoporous AAO platform is simple, easy to miniaturise, inexpensive and has great potential for development of gas sensing devices for a range of medical and environmental applications.
Hydrogen sulphide [H2S] is a colourless, corrosive, flammable and highly toxic gas commonly known through its foul odor of rotten eggs. It can be produced in sewage by bacterial breakdown, in coal mines and in the oil, chemical and natural gas industries . As an extremely toxic gas, its early detection is crucial to protect people from deadly exposures (>250 ppm) . However, recent studies showed that at lower concentrations, H2S has important biological functions . Micromolar levels of H2S have been observed in human tissues (brain and blood) suggesting that H2S is a constituent of cells, but its broader biological role is still not well understood . One of the reasons for our poor understanding is the lack of sensitive and specific analytical methods for real-time measurements of H2S in a complex biological environment. An oral malodour with major presence of H2S that arises from bacterial metabolism of amino acids and proteins is another example of biomedical determination of H2S that can be used for diagnosis of specific diseases .
Oral malodour, also known as halitosis or bad breath, is largely caused by volatile sulphur compounds [VSCs], which are produced due to bacterial degradation of proteins present in the oral cavity . In most cases, oral malodour originates as the result of microbial metabolism and degradation of proteins, especially those that contain cysteine and methionine, or peptides and aminoacids that are present in the salivary/gingival crevicular fluid or in food that is retained on the teeth. It has been previously reported that VSCs, such as hydrogen sulphide (which accounts for 80% of oral VSCs), methyl mercaptan, dimethyl sulphide and allyl mercaptan, are the major gases associated with unpleasant oral malodour [6, 7].
Diagnosis of oral malodour is conventionally performed organoleptically by a trained expert . However, such measurements are obviously variable and quantitatively limited . Several analytical methods have been devised for detection of VSCs including gas chromatography, high performance liquid chromatography, colorimetric, UV-Visible and fluorescence spectrophotometry, electrochemical (amperometric and potentiometric) methods and volumetric titrations [9–11]. However, these methods are time-consuming or require expensive equipment, skilled operators, often require a large volume of sample and cannot be used for real-time measurements. Hence, development of new methods to address these limitations for the biomedical measurement of H2S is urgently required. Optical methods are particularly attractive due to their sensitivity, simplicity, low cost, potential for in-situ measurement and ease of miniaturisation.
Reflectometric interference spectroscopy [RIfS], based on Fabry-Perot thin polymer film interference, has been effectively explored over the last two decades, mainly by the Gauglitz group, for sensing and biosensing applications including gases, hydrocarbons, herbicides, proteins and DNA [12, 13]. Studies by MJ Sailor's group showed that nanoporous structures such as porous silicon and porous anodic aluminium oxide [AAO] offer superior RIfS properties for chemical and biological sensing in comparison with thin polymer films [14–16]. The detection method is based on the reflection of white light at the top and bottom of porous structures, which generates a characteristic interference pattern with Fabry-Perot fringes . Binding of the molecular species on the pore surface induces changes of refractive index and wavelength shifts in the fringe pattern that can be easily detected and quantified . The ultimate advantage of a nanoporous AAO platform, instead of planar polymer films previously used for RIfS sensing and biosensing, is in providing a unique three-dimensional morphology of pore structures and the flexibility to be modified with specific functional groups [17–19].
Aluminium foil (0.1 mm, 99.997%) was supplied by Alfa Aesar (Ward Hill, MA, USA). Oxalic acid (Chem Supply, Pty Ltd, Adelaide, South Australia, Australia), chromium trioxide (Mallinckrodt, Inc., Miami, FL, USA), phosphoric acid (85%, BDH, VWR International Ltd., Poole Dorset, UK) and Na2S (Sigma-Aldrich Pty. Ltd., Sydney, Australia) were used as supplied. Standard gas concentrations for H2S measurements were prepared using a calibration of H2S gas mixture in air (BOC, Sydney, Australia) or by gas generated from Na2S in phosphate buffer and mixed with air. High purity water was used for all solutions preparation, as produced by sequential treatments of reverse osmosis, and a final filtering step through a 0.22-µm filter.
Preparation of nanoporous AAO
Nanoporous AAO was prepared by a two-step anodization process using 0.3 M oxalic acid as electrolyte at 0°C as previously described [20, 21, 23]. The first anodized layer of porous alumina was prepared at a voltage of 60 to 80 V and then removed using an oxide removal solution (0.2 M chromium trioxide and 0.4 M phosphoric acid). Final anodization was carried out at 60 V for 10 min in order to prepare AAO with optimal pore diameters, inter-pore distances and length.
Surface modification and structural characterisation of prepared AAO
The coating of ultra-thin metal films Au onto AAO (Au-AAO) was performed by metal vapour deposition (Emitech K975X, Quorum Technologies, Ashford, UK). The thickness of deposited films was approximately 8 nm and controlled by the film thickness monitor. The pore diameters and the thickness of the AAO porous film were determined by scanning electron microscopy [SEM] (FEI Quanta 450, FEI Company, Hillsboro, OR, USA). For cross-sectional SEM imaging, free-standing AAO substrates were prepared by removing the underlying Al. AAO samples were coated with a 5-nm Pt layer prior to SEM measurements.
Fabrication and assembly microchip sensing device
Optical setup for reflective interference measurements
Optical RIfS measurements were performed using a micro fibre optic spectrometer (Jaz-Ocean Optics, Inc., Dunedin, FL, USA). A bifurcated optical fibre with its one trunk illuminated by a tungsten lamp carried the light to the probe, and the reflected light was collected by the same probe and fed to the other trunk of the optical fibre, which at the end fed the reflected light to the spectrometer. The spot size of the light from the probe onto the AAO surface was kept around 2 mm in diameter, and all the reflective interference data were collected at a spectral range of 400 to 900 nm from the AAO film. Effective optical thickness [EOT] can be obtained by applying fast Fourier transform to the interference spectra. Fast Fourier transform from the Igor Pro (WaveMetrics, Inc., Portland, OR, USA) library was applied to finally obtain the EOT (2n eff L value in the Fabry-Perot interference fringe equation).
Real-time malodour measurements
Results and discussion
Structural characterisation of prepared AAO
Detection of H2S by RIfS Au-AAO sensor
Real-time oral malodour measurements
In conclusion, nanoporous AAO RIfS sensing for the measurement of VSCs is demonstrated. Gold-coated AAO RiFS sensor was found to have an excellent sensitivity for H2S and VSCs based on the affinity of gold surface to binding HS groups. A practical biomedical application of the RIfS Au-AAO sensor for malodour measurement was also successfully proved. The RIfS gas detection method is generic, and the coating of AAO with other gas-sensitive films can be used for the detection of specific hazardous gases. The sensing device based on a nanoporous AAO platform is simple, easy to miniaturise, inexpensive and has great potential for the development of gas sensing devices for a range of medical and environmental applications.
The authors thank the Australian Research Council (DP 0770930), the University of South Australia and the Australian National Fabrication Facility Limited (ANFF) SA node at UniSA (Ian Wark Research Institute) for the microfluidic device design and fabrication.
- Beauchamp RO, Bus JS, Popp JA, Boreiko CJ, Andjelkovich DA, Leber P: A critical review of the literature on hydrogen sulfide toxicity. CRC Crit Rev Toxicol 1984, 13: 25–97. 10.3109/10408448409029321View ArticleGoogle Scholar
- Selene C-H, Chou J, United Nations Environment Programme, International Labour Organisation, World Health Organization, Inter-Organization Programme for the Sound Management of Chemicals, International Program on Chemical Safety: Hydrogen Sulfide: Human Health Aspects. Geneva: WHO; 2003.Google Scholar
- Kabil O, Banerjee R: The redox biochemistry of hydrogen sulfide. J Biol Chem 2010, 285: 21903–21907. 10.1074/jbc.R110.128363View ArticleGoogle Scholar
- Abe K, Kimura H: The possible role of hydrogen sulphide as an endogenous neuromodulator. J Neurosci 1996, 16: 1066–1071.Google Scholar
- Krespi YP, Shrime MG, Kacker A: The relationship between oral malodor and volatile sulfur compound-producing bacteria. Otolaryngol Head Neck Surg 2006, 135: 671–676. 10.1016/j.otohns.2005.09.036View ArticleGoogle Scholar
- Rodríguez-Fernández J, Manuel Costa J, Pereiro R, Sanz-Medel A: Simple detector for oral malodour based on spectrofluorimetric measurements of hydrogen sulphide in mouth air. Anal Chim Acta 1999, 398: 23–31. 10.1016/S0003-2670(99)00381-5View ArticleGoogle Scholar
- Rodriguez-Fernandez J, Pereiro R, Sanz-Medel A: Optical fibre sensor for hydrogen sulfide monitoring in mouth air. Anal Chim Acta 2002, 471: 13–23. 10.1016/S0003-2670(02)00778-XView ArticleGoogle Scholar
- Hughes FJ, McNab R: Oral malodour--a review. Arch Oral Biol 2008, 53: S1-S7.View ArticleGoogle Scholar
- Greenman J, Duffield J, Spencer P, Rosenberg M, Corry D, Saad S, Lenton P, Majerus G, Nachnani S, El-Maaytah M: Study on the organoleptic intensity scale for measuring oral malodor. J Dental Res 2004, 83: 81–85. 10.1177/154405910408300116View ArticleGoogle Scholar
- Jayaraman S, Walia R, Alagirisamy N: Fluorescein mercuric acetate--a novel sensor for oral malodour detection. Sens Act B 2010, 148: 54–58. 10.1016/j.snb.2010.04.024View ArticleGoogle Scholar
- Alagirisamy N, Hardas SS, Jayaraman S: Novel colorimetric sensor for oral malodour. Anal Chim Acta 2010, 661: 97–102. 10.1016/j.aca.2009.11.064View ArticleGoogle Scholar
- Gauglitz G: Direct optical sensors: principles and selected applications. Anal Bioanal Chem 2005, 381: 141–155. 10.1007/s00216-004-2895-4View ArticleGoogle Scholar
- Gauglitz G: Direct optical detection in bioanalysis: an update. Anal Bioanal Chem 2010, 398: 2363–2372. 10.1007/s00216-010-3904-4View ArticleGoogle Scholar
- Lin VS-Y, Motesharei K, Dancil K-PS, Sailor MJ, Ghadiri MR: A porous silicon-based optical interferometric biosensor. Science 1997, 278: 840–843. 10.1126/science.278.5339.840View ArticleGoogle Scholar
- Alvarez SD, Li C-P, Chiang CE, Schuller IK, Sailor MJ: A label-free porous alumina interferometric immunosensor. ACS Nano 2009, 3: 3301–3307. 10.1021/nn900825qView ArticleGoogle Scholar
- Mun K-S, Alvarez SD, Choi W-Y, Sailor MJ: A stable, label-free optical interferometric biosensor based on TiO2 nanotube arrays. ACS Nano 2010, 4: 2070–2076. 10.1021/nn901312fView ArticleGoogle Scholar
- Losic D, Cole MA, Dollmann B, Vasilev K, Griesser HJ: Surface modification of nanoporous alumina membranes by plasma polymerization. Nanotechnol 2008, 19: 245704. 10.1088/0957-4484/19/24/245704View ArticleGoogle Scholar
- Jani AMM, Anglin E, McInnes S, Losic D, Shapter J, Voelcker N: Fabrication of nanoporous anodic alumina membranes with layered surface chemistry. Chem Commun 2009, 21: 3062–3064.View ArticleGoogle Scholar
- Jani AMM, Kempson IM, Losic D, Voelcker NH: Dressing in layers: layering surface functionalities in nanoporous aluminum oxide membranes. Angew Chem Int Ed 2010, 49: 7933–7937. 10.1002/anie.201002504View ArticleGoogle Scholar
- Masuda H, Fukuda K: Ordered metal nanohole arrays made by a two-step replication of honeycomb structures of anodic alumina. Science 1995, 268: 1466–1468. 10.1126/science.268.5216.1466View ArticleGoogle Scholar
- Losic D, Velleman L, Kant K, Kumeria T, Gulati K, Shapter JG, Beattie DA, Simovic S: Self-ordering electrochemistry: a simple approach for engineering nanopore and nanotube arrays for emerging applications. Aust J Chem 2011, 64: 294–301. 10.1071/CH10398View ArticleGoogle Scholar
- Losic D, Gooding JJ, Shapter JG: Influence of surface topography on alkanethiols SAMs assembled from solution and by microcontact printing. Langmuir 2001, 17: 3307–3316. 10.1021/la001462tView ArticleGoogle Scholar
- Losic D, Lillo M: Porous alumina with shaped pore geometries and complex pore architectures fabricated by cyclic anodization. Small 2009, 5: 1392–1397. 10.1002/smll.200801645View ArticleGoogle Scholar
- Ueno M, Shinada K, Yanagisawa T, Mori C, Yokoyama S, Furukawa S, Takehara S, Kawaguchi Y: Clinical oral malodor measurement with a portable sulphide monitor. Oral Diseases 2008, 14: 264–269. 10.1111/j.1601-0825.2007.01374.xView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.