Anal. Chem. 2001, 73, 462-466
Optical Sensors for Detection of Bacteria. 1. General Concepts and Initial Development Han Chuang,† Patrick Macuch,‡ and Mary Beth Tabacco*,†
Echo Technologies, Inc. and Altran Corporation, 451 D Street, Boston, MA 02210
The concept of using immobilized nucleic acid stains as detection chemistry to fabricate optical bacterial sensors is first demonstrated. SYTO 13 (a green fluorescent cell stain) is used as the molecular recognition element and fluorescent reporter in the sensor. The sensor responds to aqueous and aerosolized bacterial samples in 15 and 30 min, respectively. In addition, the sensor can discriminate a change in Pseudomonas aeruginosa (Pa) cell concentration of 1 order of magnitude or less and can detect down to 2.4 × 105 cells/mL of Pa cells. The utility of the sensor is demonstrated by monitoring the growth of a Pa cell culture over a period of 50 h. There is an immediate need for the real-time detection of microorganisms in many applications such as indoor air and potable water quality monitoring, in the food industries, in fossil and nuclear power plants, and in wastewater treatment plants. For example, the USDA estimates there are 5 million cases of foodborne illness associated with meat, poultry, and eggs and 4500 deaths/year. Estimates have placed the economic impact of foodborne illness as high as $10 billion/year.1 In another example, organisms associated with biological fouling (biofouling) and microbially induced corrosion (MIC) pose serious problems in industrial water-handling systems (i.e., corrosion in fluid conduits, mechanical parts, and other construction materials). The annual, worldwide loss caused by biofouling and MIC has been estimated to be billions of U.S. dollors.2 In recent years, the threat from biological warfare (BW) agents has become a critical issue both on the battlefield and for general public safety. BW agent-based weapons are suited to use by terrorists because they are costeffective, can potentially cause mass casualty, are easily produced, and are difficult to detect. Among the many classes of BW agents available, bacterial cells are a serious threat because they are robust, easy to deliver, and result in acute or delayed toxicity. Bacteria are considered to be potentially the most prevalent type of BW agent. Although many research efforts have been directed toward development of effective instrumental methods for the detection of microorganisms, only a few technologies show promise as realtime BW detectors.3-5 The difficulties come in part from strict requirements for sensitivity, specificity, and response time. Also, †
Echo Technologies, Inc. Altran Corp. (1) Hogan, H. Biophotonics Int. 1999, (July/Aug), 18-19. (2) Zeikus, G., Johnson, E. A., Eds. Mixed cultures in biotechnology, McGrawHill: New York, 1991; pp 341-372. ‡
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adaptation of conventional instruments such as mass spectrometers may not meet requirements for portability, ease of use, and low cost. Very elegant work has been done using antibodies and, more recently, DNA probes for detection of pathogenic microorganisms in water.6-8 Specifically in the area of foodborne pathogens, immunoelectrochemical and surface-enhanced infrared sensors have been demonstrated.9,10 These assays are however inherently complex, requiring sample preparation, addition of solvents, and washing steps. Even when these systems are automated, they necessitate the development and maintenance of fluid-handling systems, reagent reservoirs, and generate chemical waste. Also, a significant drawback to these approaches is that the exact nature of the chemical or biological contaminant must be known in advance, so that an appropriate antibody, or complementary DNA strand, can be produced and immobilized on the sensor. Detection of bacterial cells using polymerase chain reaction (PCR) is another recent technical approach. One good example is the development of a rapid, real-time PCR method for bacteria detection.11 This method combines a fluorogenic 5′nuclease assay and a spectrofluorometric thermal cycler. With this system, the time required for detection is ∼10 min, with sensitivity down to five Erwinia herbicola organisms. It is our interest to develop a simple and a widely applicable optical sensor for detection of bacteria. The idea is based on a well-known phenomenon that the fluorescence quantum yields of some nucleic acid stains significantly increase upon complexation with nucleic acids (DNA or RNA). This fluorescence signal increase can be correlated to the amount of nucleic acid present in the sample. Since all organisms contain nucleic acids, this sensor should be applicable to all bacterial species. Although fluorescent nucleic acid stains are routinely used in microscopy and cell biology, their use in optical sensing is new. This idea was first described in U.S. Patent 5,809,185, in which optical (3) Hobson, N. S.; Tothill, I.; Turner, A. P. F. Biosens. Bioelectron. 1996, 11(5), 455-477. (4) Paddle, B. M. Biosens. Bioelectron. 1996, 11(11), 1079-1113. (5) Richardson, S. D. Anal. Chem. 1999, 71(12), 181R-215R. (6) Cao, K. L.; Anderson, G. P.; Ligler, F. S.; Ezzel, J. J. Clin. Microbiol. 1995, 33, 336-341. (7) Wadkins, R. M.; Golden, J. P.; Pritsiolas, L. M.; Ligler, F. S. Biosens. Bioelectron. 1998, 13, 407-415. (8) Guschin, D. Y.; Mobarry, B. K.; Proudnikov, D.; Stahl, D. A.; Rittmann, B. E.; Mirzabekov, A. D. Appl. Environ. Microbiol. 1997, 63, 2397-2402. (9) Brewster, J. D.; Gehring, A. G.; Mazenko, R. S.; VanHouten, L. J.; Crawford, C. J. Anal. Chem. 1996, 68, 4153-4159. (10) Brown, C. W.; Li, Y.; Seelenbinder, J. A.; Pivarnik, P.; Rand, A. G.; Letcher, S. V.; Gregory, O. J.; Platek, M. J. Anal. Chem. 1998, 70, 2991-2996. (11) Belgrader, P.; Benett, W.; Hadley, D.; Richards, J.; Stratton, P.; Mariella, R., Jr.; Milanovich, F. Science 1999, 284 (5413), 449-450. 10.1021/ac000459b CCC: $20.00
© 2001 American Chemical Society Published on Web 12/28/2000
bacterial sensors were proposed.12 In this paper, we demonstrate the first semiselective bacterial sensors utilizing the SYTO 13 fluorophore immobilized on optical substrates. Our semiselective approach does not offer species or strain specificity but exhibits the potential of class specificity, such as distinguishing bacteria/ viruses, bacteria/fungi, bacteria/spores, and even live/dead and Gram positive/negative bacterial cells. The approach offers the distinct advantage of detection capability when the nature of the biological threat is not known in advance. In this paper, we present results obtained from the initial development of the bacterial sensor. These results include semiquantitative sensor response to Pa bacteria in aqueous samples and real-time detection of aerosolized bacteria using a distal-end fiber-optic sensor geometry. In addition, application of the sensor to monitoring a cell culture process is illustrated. EXPERIMENTAL SECTION Chemicals. Unless specified, all reagents were directly purchased from manufacturers without further purification. The nucleic acid fluorescent dye SYTO 13 was a product of Molecular Probes, Inc. (Eugene, OR). Phosphate-buffered saline (PBS) and tryptic soy broth (TSB) were obtained from Sigma (St. Louis, MO) and Becton Dickinson (Sparks, MD), respectively. Deionizeddistilled water was prepared from a Corning MP-6A Mega-Pure system. Pure water and PBS solutions were passed through 0.2µm filters for sterilization and stored in the dark at 4 °C. For the cell culture experiment, TSB was sterilized by autoclaving at 121 °C under 15 psi for 20 min and then refrigerated at 4 °C. Safety Precautions Regarding Use of Nucleic Acid Stains. Like acridine orange, which intercalates with nucleic acids, SYTO 13 should also be treated as a mutagen. SYTO 13 is prepared as a dimethyl sulfoxide (DMSO) solution by the manufacturer. As the manufacturer suggests, the DMSO stock solutions should be handled with particular caution because DMSO is known to facilitate the entry of organic molecules into tissues. Safe laboratory practices including double gloves, protective clothing, and eyewear should be implemented when the dye stock solution is handled. Wipers with good absorbency should be used to clean up accidental spillage. As with all other nucleic acid stains, solutions containing nucleic acid stains should be poured through activated charcoal before disposal. The charcoal must then be incinerated to destroy the dyes. Microorganisms. The cultivation, transfer, and preparation procedures for microorganisms were performed in a class II, type A laminar flow biosafety cabinet (Baker Co., Sanford, ME) using aseptic techniques. Pa cells (strain no. 10145) were a product of the American Type Culture Collection (ATCC, Rockville, MD). To prepare aqueous Pa suspension for quantitative sensor experiments, cells were grown on trypticase soy agar (TSA; Becton Dickinson, Cockeysville, MD) plates and incubated at 30 °C for 18-24 h. Pa cells were harvested from TSA, suspended in 10 mL of 0.01 M PBS buffer, and centrifuged at 5000 rpm for 15 min. The supernatant was carefully removed and the pellet resuspended in 10 mL of PBS, followed by another centrifugation-resuspension cycle. The final concentration was ∼1 × 109 cells/mL, based on an acridine orange direct count (AODC) using epifluorescence microscopy.13 (12) Mitchell, R. U.S. Patent 5,809,185, September 15, 1998.
Figure 1. The sensor geometry, optical components, and detector system. The sensing chemistry is coated on the distal end of the disposable, 1400-µm optical fiber.
In the Pa growth experiment, cells were cultured in 50 mL of TSB at 30 °C with 100 rpm of constant agitation. A 1-mL sample of culture broth was aseptically pipetted from the bulk solution at specified time intervals (Figure 3). One portion of this culture broth was serially diluted and transferred to TSA for viable cell count. The remaining portion of sample culture was pelleted by centrifuging at 5000 rpm for 15 min and washed by resuspending in 1 mL of water. One portion of the washed Pa cells was serially diluted and enumerated by the AODC method. The remaining portion of the washed Pa cells was diluted to a concentration of 3.4 × 107 cells/mL, and a 1-µL aliquot was used for sensor measurement. Preparation of Sensing Films. The procedures for preparation of the sensor films are quite simple. Two-microliter aliquots of a 50 µM SYTO 13 aqueous solution were directly pipetted onto the distal end of a series of replaceable 1400-µm-diameter optical fibers (Figure 1). The solution was allowed to dry under ambient conditions for 20 mn., resulting in a thin film of SYTO 13 on the optical fiber’s distal end. The fluorescence signal from these SYTO 13-coated optical fibers were individually acquired and then used for subsequent baseline subtraction. To regenerate the sensing surface after each single measurement, the fiber’s distal end was thoroughly cleaned three times by damped paper wipes, followed by a brief period (∼2 s) of sterilization with an oxygen-rich lighter flame. Sensor Geometry and Assembly. A 16× microscope objective was used to focus the excitation light beam on an 800-µm (core diameter) optical fiber. The fiber optic was used to carry the excitation light to the distal end and to transport the fluorescence signal to the detector. Before assembling, serial polishing steps were applied to all optical fibers, and a 0.3-µm lapping film was used for the final polishing step. In Figure 1, the sensor geometry was such that the 800-µm-diameter optical fiber end was connected to a 1400-µm-diameter optical fiber via an (13) Mittelman, M. W.; Greesey, G. G.; Hite, R. R. Microcontamination 1983, 1 (2), 32-37.
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optical connector. The larger diameter fiber presents a greater surface area for cell capture which improves sensitivity and lifetime of the sensor. However, the sensor’s optical coupling efficiency was reduced somewhat because of the mismatch in core diameter between the two coupled fibers. Instrumentation. The fiber-optic sensor geometry was based on the combination of proximal-face excitation and distal-end chemistry (Figure 1). Excitation light to the sensor was provided by a 75-W Xe arc lamp, coupled to an Acton Research Corp. (ARC, Acton, MA) model SpectraPro-150 monochromator. The excitation wavelength was set at 485 nm. The fluorescence signal was collected by a SpectraPro-300 monochromator with a photomultiplier tube (PMT) operated at 600 V. To purify the excitation beam and to eliminate stray light from the source, optical filters were used on the source monochromator exit slit (475 RDF 40, bandpass, Omega Optical, Inc., Brattleboro, VT) and the detector monochromator entrance slit (GG 495, long-pass, Schott Glass Technologies Inc., Duryea, PA). The detector entrance slit width was set at 500 µm, and the calculated resolution was 2.7 nm. Data acquisition was accomplished using a desktop computer with ARC SpectraSense data acquisition software. An ARC NCL controller box was used as the computer-spectrometer interface. The fluorescence microscope used for AODC was a Nikon Inc. (Melville, NY) Eclipse E 400 system with a 100-W mercury lamp as the excitation light source. Sample Detection. Bacterial suspensions were either pipetted directly onto the sensor distal end or collected on the distal end during aerosolization. When samples were pipetted onto the fiber surface, sensor signals were recorded 15 min after application. Although the cells begin to stain immediately, 15 min was determined to be an optimal balance between dye reaction time and solvent evaporation. Separate experiments have shown that a signal change from a sensor exposed to ∼1000 cells can be seen in less than 2 min (data not shown). Aerosolized bacterial cells were introduced to a steel bioaerosol chamber with dimensions of 10 × 5 × 2.5 in. Bacterial samples were introduced from one side of the chamber using a MRE CN24 Collison nebulizer (BGI Inc., Waltham, MA), operated at 10 psi with nitrogen as the carrier gas. The carrier gas exit port and the 1400-µm-diameter fiber-optic sensor were located on the opposite side of the chamber from the nebulizer opening. The sensor’s distal end was positioned such that it was facing the nebulizer opening offset with a distance of ∼2 in. For contamination control, the bacterial cell efflux from the chamber was captured using a 0.2-µm Teflon filter at the chamber exit port. As a safety precaution, the aerosol chamber was leak tested before each use by submerging it in water while pressurized with nitrogen to 10 psi. RESULTS AND DISCUSSION The overarching goal of our work is to build a simple, rapid, handheld optical sensor to detect air and waterborne bacteria. This study demonstrates the use of nucleic acid stains as probes to construct optical sensors for bacteria. The properties of nucleic acid stains, in fact, play a very important role in the success of our sensors. Although there are many commercially available nucleic acid stains, only a few stains are suitable for use in our sensors. We screened several nucleic acid stains based on the following criteria: (1) resistance to photobleaching and natural 464 Analytical Chemistry, Vol. 73, No. 3, February 1, 2001
Figure 2. Fluorescence signal enhancement of SYTO 13 upon reacting with Pa cells on a fiber-optic sensor. The enhancement is calculated to be 6.7 times.
degradationsto extend sensor lifetime and shelf life; (2) sufficient spectral separation (Stokes shift) between excitation and emission profilessto simplify optical filter design and selection; (3) rapid bacterial cell stainingsto reduce sensor response time; (4) visible excitation wavelengthsto avoid difficulties associated with UV excitation and to enable the use of conventional optical fibers and low-power solid-state light sources (such as LEDs) for portability; (5) high quantum yield upon interaction with bacterial nucleic acidssto maximize sensor sensitivity; (6) little or no inherent fluorescencesto reduce background interference; (7) good solubility in the sensing film matrixsto facilitate cell staining; (8) high specificity to nucleic acidssto minimize the effects of potential interferences, such as dust, lint, and other nonbiological materials. In this study, the cyanine dye SYTO 13 was found to be an excellent candidate fluorophore for detection of bacteria using optical sensors. When dissolved in water, SYTO 13 has an absorption maximum at 488 nm and an emission maximum at 509 nm. SYTO 13 is a membrane-permeable nucleic acid stain, which, in its unbound state, has a very low intrinsic fluorescence with a quantum yield of less than 0.01. The quantum yield increases to 0.4 (40-fold increase) or greater when it is bound to nucleic acids.14 This property of SYTO 13 was used to compare sensor signals, before and after exposure to a 2-µL suspension of Pa (2.4 × 107 cells/mL). Figure 2 illustrates that there is a significant fluorescence signal increase (∼6.7-fold) when the sensor is exposed to a suspension of Pa cells; this increase is much lower than the reported 40-fold increase when SYTO 13 binds to purified nucleic acids. Several factors or combinations thereof, may explain these results. First, the reported quantum yield increase is observed in aqueous bulk measurements while our study is performed on a solid-state sensor surface. The kinetics of cell staining on a relatively dry sensor surface are slower than in bulk. Second, the number of cells in the test samples was low compared to the number of dye molecules; thus, only a portion of SYTO 13 molecules are bound to nucleic acids. In addition, there is only 21 nm of spectral separation (Stokes shift) between absorption and emission spectra of SYTO 13. Therefore, it is inevitable that some stray light from the source interferes with the fluorescence detection. The ability of the sensor to monitor changes in Pa cell physiology during growth in liquid culture was also demonstrated (14) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 6th ed., Molecular Probes, Inc.:Eugene, OR, 1996; p 148.
Figure 3. A cell growth experiment comparing conventional plate counts, AODC enumeration, and sensor response. The data points on the sensor response curve is computed from integrated PMT counts between 510 and 580 nm. The sensor signal is generated from 3.4 × 104 stained Pa cells at different time points. The fluorescence signal of the SYTO 13-coated optical fiber is used for baseline subtraction from each individual sensor measurement.
(Figure 3). For easier viewing, all data sets in the plot are normalized to the same scale, by multiplying the data points with specified factors as displayed in Figure 3. As a result, the y-axis is generalized as “method response” instead of “cell number” or “integrated PMT count”. For example, the method response values of the data points on the sensor response curve represent actual, integrated PMT counts multiplying by a factor of 10-4. The unit for sensor method is “counts”, and the unit for both AODC and plate count methods is “cells/mL” (AODC and plate count data are commonly used in microbiology to determine the cell growth curve and cell viability). The data points comprising the sensor response curve are integrated fluorescence signal generated from 3.4 × 104 stained Pa cells at different physiological stages. It is shown in the plot that the sensor response is high when the cells are in the early growth phase. The sensor signal significantly drops in the middle to late growth phases (15-30 h), and levels off in the stationary phase (30 h or longer). It is known that young cells tend to have a high RNA content in a nutrient-rich environment and that level decreases as cells age.15 Furthermore, the change in cellular DNA levels is not as significant when compared to that of RNA. Because our sensor signal directly correlates to total nucleic acid concentration (DNA and RNA), the sensor response curve is actually an indication of the RNA level variation during the cell growth cycle. The plate count data in Figure 3 further corroborate this interpretation. It can be seen in Figure 3 that although the total cell number (as determined by AODC) becomes constant after 30 h of culture, the viable cell count starts to drop. These data are consistent with the reported observation that, in Escherichia coli cells, the averaged cellular RNA concentration is low when the cell viability is low.15 Therefore, with a proper calibration protocol, our sensor can possibly be used to monitor the progress of general cell culture processes in which viable cell counts can be measured in 15 min, whereas it takes a minimum of 24 h with traditional plate count methods. (15) Neidhardt, F. C.; Ingraham, J. L.; Schaechter, M. Physiology of the bacterial cells: a molecular approach; Sinauer Associates: Sunderland, MA, 1990; pp 4, 418-422.
Figure 4. Demonstration of quantitative sensor response. The excitation wavelength is 485 nm, and the sample volume is 2 µL each. Signal from the SYTO 13-coated optical fiber is used for baseline subtraction. The inset in the figure demonstrates the difference between sensor baseline (lower trace) and its response to 2.4 × 105 cells/mL Pa suspension (upper trace). In the inset, the three sharp spectral peaks around the 550- and 600-nm wavelength regions are a result of incomplete filtration of the source radiation. These spectral peaks are not present after baseline subtraction.
The quantitative sensor response as a function of cell concentration was also studied. Figure 4 illustrates that the sensor signal significantly increases with increasing cell concentration. The sensor can discriminate a change in cell concentration of 1 order of magnitude or less and can detect as few as 2 µL of 2.4 × 105 cells/mL, which corresponds to 480 bacterial cells on the sensor surface. This discrimination capability is as good as conventional microbiological enumeration techniques that can vary by ∼0.5 order of magnitude. As a result, the sensor may serve well as a very small, rapid, and semiquantitative bacteria detector for aqueous samples. Most developmental techniques for detecting microorganisms necessitate that the sample be introduced in the liquid phase, which requires a more complicated instrument design. Therefore, an additional objective of our efforts is to demonstrate the sensor operation with aerosol samples, as may be encountered in a BW attack or in a hospital environment. In one experiment, the sensor was tested for its ability to detect aerosolized Pa cells. An aqueous Pa cell suspension (5 × 107 cell/mL) was delivered into a sealed, steel reaction chamber using a BGI collison nebulizer. At the operating pressure used in the nebulizer, the mean droplet diameter is
