Packaging Sensing Cells in Spores for Long-Term Preservation of

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Packaging Sensing Cells in Spores for Long-Term Preservation of Sensors: A Tool for Biomedical and Environmental Analysis Amol Date, Patrizia Pasini, Abhishek Sangal, and Sylvia Daunert* Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055 Whole-cell sensing systems have successfully been employed for detection of various biologically and environmentally important analytes. A limitation to their use for on-field analysis is the paucity of preservation methods for long-term storage and transport. For that, we have previously developed spore-based genetically engineered whole-cell sensing systems that are able not only to maintain the activity of the sensing cells but also to preserve it for long periods of time in normal and extreme environmental conditions. Herein, we have employed these spore-based sensing systems for analysis of real samples, such as blood serum and freshwater. Spores were able to germinate in the presence of the sample matrix, and the minimum time required for the spores to germinate and generate vegetative sensing cells able to elicit a measurable response to target analytes resulted to be around 2 h. Of the two spore-based sensing systems selected to detect model analytes in real samples, one was able to detect arsenic concentrations as low as 1 × 10-7 M in freshwater and serum samples, and the other one could sense down to 1 × 10-6 M of zinc in serum. The analysis of human serum samples from healthy subjects for their zinc content proved the viability of sporebased sensing systems. The complete assays, including spore germination and analyte detection, were performed in 2.5 h or less for arsenic and zinc. Furthermore, the assay is inexpensive and simple to carry out and offers unique advantages for the incorporation of the spore-based sensing systems into portable analytical platforms, such as microfluidic devices, to be employed for on-site analysis. Genetically engineered bacterial whole-cell sensing systems are based on coupling of a specific binding event between a regulatory protein and its ligand analyte with the expression of a reporter protein within intact cells. Such systems are constructed by inserting into host cells the gene sequences of a regulatory protein, an operator/promoter (O/P) region, and a reporter gene whose transcription is under the transcriptional control of the regulatory protein and the O/P. These genes are usually carried by a DNA plasmid, which is transformed into the bacterial cells; alternatively, they can be inserted in the bacterial chromosome. An analyte permeates through the cell membrane and binds to * Corresponding author. Fax: +1 8593231069. E-mail: [email protected].

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the recognition/regulatory protein to activate the transcription of the reporter gene. Subsequent translation of the reporter mRNA produces a protein, which generates a measurable signal that is directly related to the amount of analyte. With the choice of the appropriate regulatory and reporter proteins, these sensing systems can yield quantitative information about the analyte of interest in in vitro as well as in vivo settings. Additionally, since the target analyte needs to be uptaken by the sensing cells to induce a response, only the bioavailable amount of analyte is measured. This feature is unique to whole-cell sensing systems, which contributed to the appeal that these analytical tools gained in several fields of bioanalysis, such as environmental monitoring, drug screening, and clinical analysis. Bacterial whole-cell biosensing systems have been developed for the detection of sugars, drugs, quorum sensing molecules, various toxic metals, such as mercury, arsenic, cadmium, and several organic pollutants.1-7 These bacterial sensing systems can selectively, sensitively, and rapidly detect very low levels of analytes. Furthermore, the potential of these sensors to be integrated into portable devices, including attachment onto optic fiber tips, incorporation into miniaturized microfluidics platforms, and immobilization on paper strips, among others, makes them attractive for on-field analysis.2,8,9 However, limitations to the on-site use of these sensing systems are posed by their short shelf life and the requirement of specific growth and storage conditions. Several approaches with varying degrees of success, including freeze- and vacuum-drying, continuous culture and encapsulation in organic as well as inorganic polymers, have been proposed to address these major chal(1) Galluzzi, L.; Karp, M. Comb. Chem. High Throughput Screening 2006, 9, 501–514. (2) Rothert, A.; Deo, S. K.; Millner, L.; Puckett, L. G.; Madou, M. J.; Daunert, S. Anal. Biochem. 2005, 342, 11–19. (3) Shetty, R. S.; Deo, S. K.; Shah, P.; Sun, Y.; Rosen, B. P.; Daunert, S. Anal. Bioanal. Chem. 2003, 376, 11–17. (4) Turner, K.; Xu, S.; Pasini, P.; Deo, S.; Bachas, L.; Daunert, S. Anal. Chem. 2007, 79, 5740–5745. (5) Daunert, S.; Barrett, G.; Feliciano, J. S.; Shetty, R. S.; Shrestha, S.; SmithSpencer, W. Chem. Rev. 2000, 100, 2705–2738. (6) Kumari, A.; Pasini, P.; Deo, S. K.; Flomenhoft, D.; Shashidhar, H.; Daunert, S. Anal. Chem. 2006, 78, 7603–7609. (7) Shetty, R. S.; Ramanathan, S.; Badr, I. H. A.; Wolford, J. L.; Daunert, S. Anal. Chem. 1999, 71, 763–768. (8) Stocker, J.; Balluch, D.; Gsell, M.; Harms, H.; Feliciano, J.; Daunert, S.; Malik, K. A.; vanderMeer, J. R. Environ. Sci. Technol. 2003, 37, 4743– 4750. (9) Polyak, B.; Bassis, E.; Novodvorets, A.; Belkin, S.; Marks, R. S. Sens. Actuators, B: Chem. 2001, 74, 18–26. 10.1021/ac1007865  2010 American Chemical Society Published on Web 06/18/2010

lenges.10 Another issue is the actual applicability of these preserved cells to the analysis of real samples, given the potential interference of the sample matrix components with the sensing system response. For that, we have previously developed a new method for maintaining the viability and analytical performance of whole-cell biosensing systems, which allows for their long-term preservation and storage under conditions as simple as room temperature.11 Specifically, this method is based on the use of spore-forming bacteria as host microorganisms for the development of wholecell sensing systems. Spores of these bacterial cells are then generated as robust preservation, storage, and transport vehicles of the sensing cells. Such spores are subsequently converted to fully active vegetative sensing cells when needed. Herein, we have demonstrated the feasibility of directly applying spore-based sensing systems to the detection of target analytes in biological and environmental samples. To that end, we have developed analytical tools for quantitative analysis of arsenic and zinc, respectively, in real samples, such as blood serum and freshwater. EXPERIMENTAL SECTION Reagents. Sodium phosphate (monobasic), sodium arsenite, sodium bicarbonate, calcium sulfate, magnesium sulfate, potassium chloride, zinc chloride, ampicillin, erythromycin, and human serum (from clotted human male whole blood, sterile-filtered, mycoplasma tested, virus tested) were obtained from SigmaAldrich Corp. (St. Louis, MO). LB broth was obtained from Difco (Sparks, MD). All chemicals were reagent grade or better and were used as received. The chemiluminescence assay kit for β-galactosidase was purchased from Promega (Madison, WI) and used as suggested by the manufacturer. All solutions were prepared using reverse osmosis water (Milli-Q Water Purification System, Millipore, Bedford, MA). Apparatus. Chemiluminescence and fluorescence measurements were performed in microtiter plates (Corning Inc., Corning, NY) on a Polarstar Optima microplate luminometer from BMG Labtech (Durham, NC). All measurements were conducted at room temperature unless specified otherwise. All luminescence intensities reported are the average of a minimum of three replicates and are expressed in relative light units (RLU). Bacterial Sensing Strains and Plasmids. The sensing system for arsenic consisted of Bacillus subtilis cells transformed with plasmid pMUTinT3 to generate the ars-23 B. subtilis strain.12 The plasmid contains the lacZ gene encoding for β-galactosidase, which is under the control of the ArsR regulatory protein through the ars operon promoter. The plasmid was kindly provided by Dr. Tsutomu Sato from Tokyo University of Agriculture and Technology, Japan. The zinc sensing system consisted of Bacillus megaterium cells transformed with the plasmid pSD202. The pSD202 vector contains the O/P region of the smt operon, the smtB gene, and the egfp gene encoding for enhanced green fluorescent protein (EGFP) placed downstream under control of the smt operon promoter.11 Bacterial Spores. Spores of bacterial sensing cells, i.e., the arsenic ars-23 B. subtilis strain and the zinc B. megaterium strain (10) Bjerketorp, J.; Ha˚kansson, S.; Belkin, S.; Jansson, J. K. Curr. Opin. Biotechnol. 2006, 17, 43–49. (11) Date, A.; Pasini, P.; Daunert, S. Anal. Chem. 2007, 79, 9391–9397. (12) Sato, T.; Kobayashi, Y. J. Bacteriol. 1998, 180, 1655–1661.

bearing plasmid pSD202, were prepared by using standard protocols and media, as described elsewhere.13 Briefly, spores were generated by placing bacterial cells in sporulation medium in an orbital shaker (model no. 4518, Forma Scientific, Marietta, OH) at 37 °C, 250 rpm for 4 days. Aliquots of 1.5 mL of these sporulated cells were transferred to 1.5 mL microcentrifuge tubes and centrifuged at 10 000 rpm for 1 min using a 5417C centrifuge (Eppendorf, Westbury, NY). The supernatant was discarded, and the spore pellet was then stored at room temperature until needed. Spore Germination Study for ars-23 B. subtilis. Standard solutions of sodium arsenite at concentrations ranging from 1 × 10-3 to 1 × 10-9 M were prepared in reverse osmosis water by serial dilution of freshly prepared 1 × 10-2 M stock solution of sodium arsenite. A volume of 750 µL of LB broth was added to each of the microcentrifuge tubes containing the spore pellet, prepared as described above, to resuspend the spores and initiate germination. In order to indirectly estimate the number of spores per volume unit employed, the optical density at 600 nm (OD600 nm) of the obtained spore suspension was measured. Measurements were performed on volumes of 45 µL in triplicate using a microtiter plate reader (Polarstar Optima, BMG Labtech, Durham, NC) and OD600 nm values of 0.60-0.65 were obtained. Aliquots of 45 µL of LB spore suspension were transferred to the wells of a 96-well microtiter plate. The spores were then incubated in an orbital shaker for 1, 2, and 3 h at 37 °C, 250 rpm for germination. A volume of 5 µL of arsenite solutions at various concentrations and reverse osmosis water as a blank was added in triplicate to the germinating spores/ vegetative cells after the various times allowed for germination. The microtiter plate was then incubated in the orbital shaker at 37 °C, 250 rpm for 1 h. Next, 50 µL of the chemiluminescent substrate was added to each of the wells and incubated at 37 °C, 250 rpm for 30 min. The luminescence signal was measured at 540 nm using the microplate luminometer. After evaluation of the germination time required to obtain responsive sensing cells, the assay was performed by directly incubating the spores with the analyte standard solutions. Specifically, aliquots of 45 µL of LB spore suspension were transferred to the wells of a 96-well microtiter plate. A volume of 5 µL of arsenite solutions at various concentrations (1 × 10-3 to 1 × 10-9 M) and reverse osmosis water as a blank was immediately added in triplicate to the spore suspensions. The assay was then performed as described above, with a 2 h incubation time of the spores with the analyte. Spore Germination Study for B. megaterium Containing Plasmid pSD202. Standard solutions of zinc chloride at concentrations ranging from 1 × 10-3 to 1 × 10-8 M were prepared in reverse osmosis water by serial dilution of a freshly prepared 1 × 10-2 M stock solution of zinc chloride. Spores of B. megaterium containing plasmid pSD202 were resuspended in LB broth using the same protocol as above. Aliquots of 45 µL of LB spore suspension were then transferred to the wells of a 96-well microtiter plate. A volume of 5 µL of 1 × 10-3 M zinc chloride and reverse osmosis water as a blank was immediately added in triplicate to the spore suspensions and incubation was carried out in an orbital shaker at 37 °C, 250 rpm. Fluorescence (13) Harwood, C. R.; Cutting, S. M. Molecular Biological Methods for Bacillus; Wiley: New York, 1990.

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UT), a national clinical reference laboratory (http://www.aruplab.com).

measurements were taken every 20 min up to 230 min to define the optimum germination/analyte incubation time. In another experiment to obtain a dose-response curve for zinc, 5 µL of zinc solutions at various concentrations and reverse osmosis water as blank was added in triplicate to the wells of a microtiter plate containing 45 µL/well of spore suspension, and fluorescence measurements were performed after 150 min. Fluorescence measurements for both experiments were carried out using the microplate luminometer, with the excitation wavelength set at 480 nm and emission wavelength at 510 nm. Analysis of Freshwater and Human Blood Serum Samples. Freshwater was prepared from the following ingredients (milligrams per liter): NaHCO3 (384), CaSO4 · H2O (240), MgSO4 (240), KCl (16) in reverse osmosis water, adjusted to pH 7.14 Analyte solutions of concentrations ranging from 1 × 10-3 to 1 × 10-9 M were prepared in simulated freshwater as well as commercially available serum by serial dilutions of freshly prepared 1 × 10-2 M stock solution of sodium arsenite in reverse osmosis water. A total of 23 water samples of various origins, including, tap water from city water systems, tap water from private water wells, and natural surface water (lakes, ponds, rivers, creeks) were collected from several locations in Kentucky and Indiana. Specifically, we obtained 8 tap water samples from city water supply systems (Frankfort, KY; Erlanger, KY; Barbourville, KY; Noblesville, IN; Union, KY; Lexington, KY; Owensboro, KY; Fancy Farm, KY); 5 tap water samples from private water wells (Harned, KY; Barbourville, KY; Tripton, IN; Leesburg, IN; Georgetown, KY); 5 water samples from lakes or ponds (Harned, KY (2); Leesburg, IN; Georgetown, KY; Fancy Farm, KY), and 5 water samples from rivers or creeks (Covington, KY (2); Barbourville, KY; Owensboro, KY; Benton, KY). The arsenic spiked freshwater and serum samples as well as the collected water samples were assayed as described above for the arsenite dose-response curve. The tap and surface water samples were also tested for arsenic by a graphite furnace atomic absorption spectrometry (GFAAS) method, which is reported to have a detection limit of 5 ppb. This analysis was performed at the University of Kentucky Environmental Research Training Laboratories (ERTL) (http://ertl.uky.edu). Similarly, analyte solutions of concentrations ranging from 1 × 10-3 to 1 × 10-8 M were prepared in commercially available human serum by serial dilutions of freshly prepared 1 × 10-2 M stock solution of zinc chloride in reverse osmosis water. The prepared zinc spiked serum samples as well as serum samples obtained from healthy volunteers were assayed as described above for the zinc dose-response curve. For calculation of the zinc concentrations in healthy volunteers’ serum samples, a variable slope sigmoidal dose-response curve was employed (GraphPad Prism 4, GraphPad Software, Inc., San Diego, CA). The sample signal values were corrected for the serum background fluorescence, and the equation of the dose-response curve was then employed to correlate the corrected signals to the corresponding concentrations of zinc. The zinc concentrations in serum samples from healthy volunteers were also determined by inductively coupled plasma/mass spectrometry (ICPMS). This analysis was performed at ARUP Laboratories (Salt Lake City,

RESULTS AND DISCUSSION The necessity of simple, fast, sensitive, and specific/selective analytical methods has been one of the premier reasons for wholecell biosensing systems to draw interest in various fields of bioanalysis. On the other hand, major challenges exist that have hindered the application of these systems out of the laboratory and their exploitation on a commercial basis, namely, their shelf life and transportability. In an earlier study, we developed a new cost-effective and easy method for long-term preservation of such whole-cell bacterial sensing systems that addresses these limitations by taking advantage of the durability and ruggedness of bacterial spores. In fact, spores are known to be able to be quiescent and preserve bacterial DNA for long periods of time even in harsh conditions such as extreme temperatures and humidity/drought.15 This method should allow not only to prolong the viability and activity of whole-cell sensing systems, in normal and extreme environmental conditions, but also to package them in a convenient manner so that they can be used more effectively in the field, thus facilitating on-site applications. Specifically, we have developed spore-based sensing systems for detection of arsenic and zinc, respectively, and demonstrated the ability of spores to preserve the analytical characteristics of whole-cell sensing systems for extended periods of time. We initially showed that the bacterial sensing systems for arsenic and zinc could not only be stored for at least 6 and 8 months, respectively, at room temperature,butcouldalsobesubjectedto3sporulation-germination cycles, where the cells alternated between dormant and active states, while maintaining their analytical parameters.11 We subsequently proved that the sensing systems retained their analytical performance upon storage for up to 24 months at room temperature as well as for up to 12 months in extreme temperature and humidity/drought conditions (manuscript in preparation). This method not only provides a way to stabilize the biosensing systems with minimum storage requirements but also opens up the possibility of application of such sensors for on-field analysis. To be effective for on-site detection of analytes, the sensors should also be fast and easy to use. While these features can be found in whole-cell sensing systems, the quality of spores to germinate as soon as they sense nutrients in their environment has not been explored in the context of spore-based sensing systems. Rapid germination of spores to vegetative fully active cells is a key requirement for spore-based sensing systems to be successfully employed in on-site analysis of real samples. To that end, we evaluated the germination time required for the spores to convert to vegetative cells and respond to the analytes of interest in order to carry out the assay in the shortest time possible. The arsenic sensing spores were allowed to germinate for various periods of time (1, 2, and 3 h). The spore-derived vegetative cells were then exposed to various concentrations of arsenite, and chemiluminescence was measured after addition of the chemiluminescent substrate for β-galactosidase. This substrate employs a coupled enzyme reaction in which 6-O-β-galactopyranosyl luciferin is cleaved by β-galactosidase to yield free luciferin. The luciferin produced is then used in a reaction catalyzed by luciferase

(14) Griffin, B. A.; Jurinak, J. J. Soil Sci. 1973, 116, 26–30.

(15) Driks, A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3007–3009.

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Figure 1. Dose-response curves of the spore-based B. subtilis arsenic sensing system after germination of spores for 1 ([), 2 (9), and 3 (2) h. Data shown are the average ( 1 standard deviation (n ) 3).

to generate a luminescent signal proportional to the amount of β-galactosidase expressed, which is, in turn, proportional to the amount of arsenite present. The germination study showed that a 2 h germination was the minimum time required for the spores to generate viable cells able to elicit a response to arsenite (Figure 1). A detection limit of 1 × 10-7 M and a dynamic range of 1 × 10-7 to 1 × 10-4 M for arsenite were observed. The detection limit was defined as the lowest tested concentration of the analyte that elicited a signal that was higher than the blank average signal plus 3 times the standard deviation of the blank. No significant analyte-dependent β-galactosidase activity could be detected at time lower than 2 h. This is consistent with previous observations that germinating spores cannot transform into vegetative cells and resume their full metabolic activities in less than 2 h.16 Subsequently, spores were directly incubated with arsenite standard solutions allowing germination to occur in the presence of the analyte. An overall incubation time of 2 h, including spore germination and expression of the reporter protein, was sufficient to achieve a detection limit of 1 × 10-7 M, with a dynamic range of 1 × 10-7 to 1 × 10-4 M. This ability of spores to germinate and originate active/responsive cells in a short period of time adds to the merits of using spores as storage vehicles for whole-cell sensing systems. Since a dose -response curve could be obtained at the incubation time of 2 h for arsenic sensing spores, this time was used for all the next experiments with this sensing system. The fluorescent zinc sensing B. megaterium spores were also evaluated for the minimum time required to elicit a response to 1 × 10-4 M of zinc. This concentration was chosen because previous characterization of the whole-cell sensing system showed the highest response with 1 × 10-4 M of zinc. Since fluorescence detection does not require any substrate addition, germination and response to the analyte could be monitored over time on the same batch of spores, without a need for setting several spore suspensions subjected to various germination times. The fluorescence was measured at 20 min intervals starting at 10 min after the addition of the analyte to the spores. A significant increase in fluorescence was observed from 110 min up to 230 min of incubation with the analyte (Figure 2). Upon these results, for studies with various concentrations of zinc, (16) Rousseau, P.; Halvorson, H. O.; Bulla, L. A., Jr.; Julian, G. S. J. Bacteriol. 1972, 109, 1232–1238.

Figure 2. Time-dependent response of the spore-based B. megaterium zinc sensing system to 1 × 10-4 M zinc. Data shown are the average ( 1 standard deviation (n ) 3).

Figure 3. Dose-response curves of the spore-based B. megaterium zinc sensing system obtained with zinc standard solutions ([) and serum samples spiked with various concentrations of zinc (9). Fluorescence measurements were performed at a germination/analyte incubation time of 150 min. Data shown are the average ( 1 standard deviation (n ) 3).

the spores were incubated with the analyte for 150 min. A dose-response curve was obtained, which demonstrated a detection limit of 1 × 10-6 M with a dynamic range of 1 × 10-6 to 1 × 10-4 M (Figure 3). The same incubation time was also employed for all the next experiments with zinc sensing spores. It is noteworthy that the above assays were carried out in a microtiter plate with a volume of 50 µL of reaction mixture (5 µL of standard/sample + 45 µL of spore suspension). This is a significant decrease in volume when compared to conventional assays in culture tubes where reaction mixture volumes are in the order of milliliters. The use of the microtiter plate format also allowed speeding up the steps of assay preparation and execution. The complete assays from beginning to end, including spore germination and analyte detection, were performed in 2.5 h or less for arsenic and zinc. Importantly, the employment of sporebased sensing systems and their direct incubation with analytes in the presence of germination/growth media eliminated the need for overnight culture of the bacterial sensing cells that is generally required for sensing cells traditionally stored in glycerol stocks at -80 °C. While overnight growth of the sensing cells could also be avoided using other strategies for cell preservation aimed at enhancing on-site application of whole-cell sensing systems, such as, freeze- or liquid-drying of the sensing cells, these methods failed in preserving the cells for more than a few weeks or a few Analytical Chemistry, Vol. 82, No. 14, July 15, 2010

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Figure 4. Dose-dependent responses of the spore-based B. subtilis arsenic sensing system obtained with various arsenite concentrations in different matrixes, i.e., buffer, freshwater and human blood serum. The relative standard deviation for the data shown is below 12%.

months and their performance in adverse storage/environmental conditions has not been reported.8,17 Overall, our results showcased two distinct advantages of spore-based sensing systems, namely, short assay time and use of microliter volumes of samples and reagents. Both of these parameters are critical for assay miniaturization and incorporation of sensing systems into portable analytical platforms, e.g., microfluidics devices. The next step in our study was to evaluate the possibility of employing spore-based whole-cell sensing systems for the direct analysis of biological and environmental samples. It should be pointed out that while the analytes selected for this work served as model analytes to prove the viability of spore-based sensors, their detection in certain types of samples is actually relevant. Specifically, arsenic is a naturally occurring element widely distributed in the earth’s crust. Accidental exposure to arsenic may occur through ingestion of arsenic contaminated food and drinking water or inhalation. Arsenic is a well-characterized environmental contaminant with recognized toxic effects on humans that lead to vascular diseases, dermatitis, and cancer, among others.18 Although most of the arsenic introduced in the body is quickly excreted through urine, it can be found in blood in various forms, such as methylated arsenic and inorganic arsenic.19 Therefore, the analysis of serum samples for their arsenic content may be considered an effective way for detection and monitoring of exposure to arsenic. Moreover, arsenic contamination of drinking water, due to natural occurrence or human activities, such as mining and other industrial activities,20 is a major concern in vast, highly populated areas of the world. Arsenic detection in drinking water with simple and cost-effective methods, which are particularly appealing in developing countries, has also been a challenge that is yet to be addressed. Therefore, the detection of arsenic in such matrixes as serum and water is very important for early detection and remediation measures. To date, several spectroscopic methods have been employed for detection of arsenic.21-23 However, most of these methods require expen(17) Gu, M. B.; Choi, S. H.; Kim, S. W. J. Biotechnol. 2001, 88, 95–105. (18) Smith, A. H.; Hopenhayn-Rich, C.; Bates, M. N.; Goeden, H. M.; HertzPicciotto, I.; Duggan, H. M.; Wood, R.; Kosnett, M. J.; Smith, M. T. Environ. Health Perspect. 1992, 97, 259–267. (19) Zhang, X.; Cornelis, R.; De Kimpe, J.; Mees, L.; Lameire, N. Clin. Chem. 1997, 43, 406–408. (20) Bech, J.; Poschenrieder, C.; Llugany, M.; Barcelo´, J.; Tume, P.; Tobias, F. J.; Barranzuela, J. L.; Va´squez, E. R. Sci. Total Environ. 1997, 203, 83–91. (21) Hung, D. Q.; Nekrassova, O.; Compton, R. G. Talanta 2004, 64, 269–277. (22) Melamed, D. Anal. Chim. Acta 2005, 532, 1–13.

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sive lab instrumentation and trained personnel. Therefore, there is a need for simple and effective methods for detection of arsenic in environmental and biological samples that can yield results in a short time. Several commercial field test kits are available for determination of arsenic in well water. However, the reliability and sensitivity of such testing kits is reported to be questionable.24 Hence, to further demonstrate the applicability of our spore-based sensing systems for on-field analysis in real samples, we evaluated their ability to detect arsenic in different matrixes. For that, serum samples spiked with various arsenite concentrations were employed to evaluate the analytical performance of the arsenic sporebased sensing system. A detection limit of 1 × 10-7 M of arsenite and a dynamic range of 1 × 10-7 to 1 × 10-4 M were achieved in serum, which were consistent with those obtained in the absence of biological matrix. The results of these experiments are reported in Figure 4. Decreased chemiluminescence signals were observed for the serum samples spiked with arsenite, as compared to the signals produced by the same analyte concentrations in the absence of the biological matrix. Dilution of serum helped in reducing this effect (data not shown). It is believed that part of the arsenic may bind to proteins in the serum, thus making the analyte less available for diffusion into the sensing cells. However, this did not negatively affect the analytical performance of the sensing spores in serum. Freshwater samples were also analyzed using the arsenic sensing ars-23 spores. For that, arsenic spiked freshwater samples were prepared by dissolving various concentrations of arsenic in simulated freshwater, prepared as described in the Experimental Section. Arsenic concentrations as low as 1 × 10-7 M could be detected. Freshwater samples did not exhibit any matrix effect, as shown by no significant changes in signal intensities as well as detection limit, sensitivity (slopes of the linear part of the dose-response curves), and dynamic range when compared to arsenic standard solutions in reverse osmosis water (Figure 4). No detectable levels of arsenic were found in any of the tap and surface water samples collected in Kentucky and Indiana. These results were confirmed by GFAAS analysis. (23) Dai, X.; Nekrassova, O.; Hyde, M. E.; Compton, R. G. Anal. Chem. 2004, 76, 5924–5929. (24) Rahman, M. M.; Mukherjee, D.; Sengupta, M. K.; Chowdhury, U. K.; Lodh, D.; Chanda, C. R.; Roy, S.; Selim, M.; Quamruzzaman, Q.; Milton, A. H.; Shahidullah, S. M.; Rahman, M. T.; Chakraborti, D. Environ. Sci. Technol. 2002, 36, 5385–5394.

The next analyte employed as a model in this study, zinc, is an essential element required for normal metabolic functions of many proteins in humans. Deficiency of zinc can lead to various health issues such as abnormal growth, reproductive failure, and impaired wound healing and immune system.25 Furthermore, higher levels of zinc in serum have been shown to be present in patients with Alzheimer’s disease.26 Therefore, the analysis and detection of zinc in serum is important. Accurate measurement of this analyte by means of voltametric and spectrometric methods27,28 requires laboratory analysis, sophisticated and expensive techniques and facilities, as well as trained staff not easily available or affordable in many parts of the world. For that, we decided to evaluate the effectiveness of our spore-based zinc sensing system for detection of zinc in serum samples spiked with various concentrations of zinc. The sensing system was able to detect zinc in serum samples down to 1 × 10-6 M, while the range of detection resulted to be 1 × 10-6 to 1 × 10-4 M of zinc, similar to experiments performed in the absence of biological matrix (Figure 3). An overall increase in the fluorescence signals observed with serum samples may be attributed to the fluorescent emission of proteins in serum that are excited at the EGFP reporter protein excitation wavelength. Moreover, the presence of zinc in the human serum employed for the spiking studies may also contribute to the increase in fluorescence observed. These results prompted us to validate our method on human serum samples collected from healthy subjects. Analysis of 8 individual serum samples revealed zinc concentrations ranging from 12-16 µM, which are within previously reported normal zinc values in human serum;29 furthermore, these results were confirmed by ICPMS analysis of the serum samples (Table 1). Importantly, we showed that spores were able to germinate in the presence of various sample matrixes. For both spore-based sensing systems, the assay timing and analytical characteristics achieved with analyte standard solutions were maintained when the sensors were applied to the analysis of real samples, namely, serum and freshwater. CONCLUSIONS The use of spores not only provides an avenue for preservation of whole-cell sensing systems but also facilitates their application (25) Hambidge, M. J. Nutr. 2000, 130, 1344S–1349. (26) Rulon, L.; Robertson, J.; Lovell, M.; Deibel, M.; Ehmann, W.; Markesbery, W. Biol. Trace Elem. Res. 2000, 75, 79–85. (27) Guo, Z.; Feng, F.; Hou, Y.; Jaffrezic-Renault, N. Talanta 2005, 65, 1052– 1055. (28) Wah Fong, B. M.; Siu, T. S.; Kit Lee, J. S.; Tam, S. Clin. Chem. Lab. Med. 2009, 47, 75–78. (29) Ghayour-Mobarhan, M.; Taylor, A.; New, S.; Lamb, D.; Ferns, G. Ann. Clin. Biochem. 2005, 42, 364–375.

Table 1. Zinc Concentrations Detected in Serum Samples from Eight Healthy Subjects by Employing the Spore-Based B. megaterium Zinc Sensing System and Inductively Coupled Plasma Mass Spectrometry human serum sample

spore sensing system zinc (× 10-5 M)

ICPMS zinc (× 10-5 M)

subject 1 subject 2 subject 3 subject 4 subject 5 subject 6 subject 7 subject 8

1.30 ± 0.10 1.64 ± 0.05 1.60 ± 0.10 1.37 ± 0.08 1.16 ± 0.05 1.54 ± 0.09 1.29 ± 0.06 1.64 ± 0.10

1.02 1.16 1.22 1.18 1.33 1.32 1.12 1.41

to on-field analysis of real samples. Herein, we have taken advantage of the ability of spores to germinate rapidly to perform assays in a time efficient manner, namely, in 2.5 h or less, including spore germination and analyte detection and quantification. Furthermore, we employed the sensing systems for detection of arsenic and zinc, respectively, in spiked freshwater and serum samples. The data obtained showed that the assays could be performed in both matrixes efficiently, without loss of analytical performance of the sensing systems, and directly from dormant spores to quantitative results in samples in a short time. These features of spore-based sensing systems will enhance their integration into miniaturized portable platforms, such as, micrototal analysis systems (µ-TAS). Upon miniaturization and incorporation into an appropriate analytical platform, spore-based genetically engineered whole-cell sensing systems have the potential to be developed into a rapid, high-throughput, fieldportable method for the detection of target analytes in both environmental and biological samples. ACKNOWLEDGMENT This work was supported by the Superfund Research Program (SRP) of the National Institute of Environmental and Health Sciences (NIEHS), Grant P42ES07380, the National Science Foundation (NSF), Grant CHE-0718844, and the United StatesIsrael Binational Agricultural Research and Development (BARD) Fund, Grant US-3864-06. We would like to thank Dr. Tsutomu Sato (Tokyo University of Agriculture and Technology, Japan) for kindly providing the plasmid pMUTinT3 and the bacterial strain B. subtilis ars-23. Received for review March 26, 2010. Accepted June 9, 2010. AC1007865

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