Anal. Chem. 2005, 77, 2683-2689
Articles
Ultrasensitive Reporter Protein Detection in Genetically Engineered Bacteria Mona Wells,*,† Michael Go 1 sch,† Rudolf Rigler,† Hauke Harms,‡ Theo Lasser,† and Jan Roelof van der Meer§
Laboratories of Soil Science and Biomedical Optics, Swiss Federal Institute of Technology (EPFL), CH-1015, Lausanne, Switzerland, Department of Microbiology, UFZ Centre for Environmental Research, D-04318, Leipzig, Germany, and Department of Fundamental Microbiology, University of Lausanne, CH-1015, Lausanne, Switzerland
We demonstrate the use of laser-induced fluorescence confocal spectroscopy to measure analyte-stimulated enhanced green fluorescent protein (egfp) synthesis by genetically modified Escherichia coli bioreporter cells. Induction is measured in cell lysates and, since the spectroscopic focal volume is approximately the size of one bioreporter cell, also in individual live bacteria. This is, to our knowledge, the first ever proof-of-concept work utilizing instrumentation with single-molecule detection capability to monitor bioreporter response. Although we use arsenic inducible bioreporters here, the method is extensible to gfp/egfp bioreporters that are responsive to other substances.
The development of sensors for both routine applications and research has been motivated by the need for detection of an increasingly vast array of compounds in complex matrixes such as food, biomedical samples (e.g., blood or tissue), and environmental samples (e.g., soils or sediments); the advent of biotechnology has expanded this even further, particularly in the area of genetically modified bioreporter bacteria,1-4 also called whole-cell biosensors. Live whole-cell bioreporter sensing elements offer greater scope than purely chemical sensing elements for information on, for example, a compound’s bioavailability, affect on living systems, and synergistic or antagonistic behavior toward biota in mixtures.5,6 Also, bioreporters have the advantage of * Corresponding author. Current address: Department of Chemistry, Foster Hall, Tennessee Technological University, Cookeville, TN 38505. Tel.: 931 372 6521. Fax: 931 372 3434. E-mail:
[email protected]. † Swiss Federal Institute of Technology. ‡ UFZ Centre for Environmental Research. § University of Lausanne. (1) Belkin, S. Curr. Opin. Microbiol. 2003, 6, 206-212. (2) Daunert, S.; Barrett, G.; Feliciano, J. S.; Shetty, R. S.; Shrestha, S.; SmithSpencer, W. Chem. Rev. 2000, 100, 2705-2738. (3) Hansen, L. H.; Sorensen, S. J. Microb. Ecol. 2001, 42, 483-494. (4) Kohler, S.; Belkin, S.; Schmid, R. D. Fresenius J. Anal. Chem. 2000, 366, 769-779. (5) Rensing, C.; Maier, R. M. Ecotoxicol. Environ. Saf. 2003, 56, 140-147. (6) Tauriainen, S. M.; Virta, M. P. J.; Karp, M. T. Water Res. 2000, 34, 26612666. 10.1021/ac048127k CCC: $30.25 Published on Web 03/26/2005
© 2005 American Chemical Society
Scheme 1. Generalized Mode of Bioreporter Response to a Target Compound
tailored protein engineering,2,7 as recently demonstrated by the successful design, construction, and integration of a high-affinity TNT binding protein into bacteria.7 Bioreporter function typically depends on a target compound passing through the cell membrane and binding to a regulatory protein; this then activates transcription of the reporter gene, and subsequent translation of the reporter mRNA results in the production of a spectroscopically active reporter molecule as depicted in Scheme 1. Detection of reporter molecules is most often achieved with standard colorimetry, fluorometry, or luminometry, wherein instrument response is related to analyte exposure concentrationsi.e., the chromophore or fluorophore itself is never quantified. Method detection limits (MDLs) from such detection schemes, when reported, are generally higher than MDLs from standard instrumental techniques of chemical analysis having more sophisticated detection capability (e.g., mass spectrometric detection),4,8-14 though notable exceptions exist. (7) Looger, L. L.; Dwyer, M. A.; Smith, J. J.; Hellinga, H. W. Nature 2003, 423, 185-190. (8) Hansen, L. H.; Sorensen, S. J. FEMS Microbiol. Lett. 2000, 193, 123-127. (9) Tauriainen, S.; Virta, M.; Chang, W.; Karp, M. Anal. Biochem. 1999, 272, 191-198. (10) Corbisier, P.; van der Lelie, D.; Borremans, B.; Provoost, A.; de Lorenzo, V.; Brown, N. L.; Lloyd, J. R.; Hobman, J. L.; Csoregi, E.; Johansson, G.; Mattiasson, B. Anal. Chim. Acta 1999, 387, 235-244. (11) Applegate, B. M.; Kehrmeyer, S. R.; Sayler, G. S. Appl. Environ. Microbiol. 1998, 64, 2730-2735.
Analytical Chemistry, Vol. 77, No. 9, May 1, 2005 2683
Here we report a promising advance in bioreporter signal detection strategy that utilizes laser-induced fluorescence confocal spectroscopy (LIF-CS).15 Using LIF-CS offers several distinct advantages over other techniques, including lower MDLs and the capability for performing intracellular analysis. Additionally, using LIF-CS, the number of reporter molecules in the focal volume is obtained directly in experimental situations wherein a fluorescence autocorrelation curve is obtained; other detection strategies in common use rely upon inference of the amount of reporter molecule present (e.g., in epifluorescent microscopy, response is measured in average gray value and in steady-state fluorometry it is measured in relative fluorescence units). The instrumental configuration used here utilizes a tightly focused laser beam to produce a ∼1-fL spectroscopic volume element and has a high signal-to-noise ratio, resulting in singlemolecule detection capability.16-22 For experimental conditions in which fluorophores freely diffuse in and out of the focal volume (e.g., lysates), a fluorescence autocorrelation curve can be obtained, which in turn directly yields the number of fluorophore molecules per focal volume, or the actual fluorophore concentration.15,23,24 This eliminates the need to relate analyte exposure concentration to arbitrary units of instrumental response. Green fluorescent protein (gfp) is a reporter molecule that is well suited to LIF-CS and also does not suffer from the complication of requiring substrate or cofactors to produce a response (as other reporter molecules do), though we use so-called enhanced gfp (egfp) here as a result of its superior stability.4,25,26 Because of the low sample volume benefit of LIF-CS, we are able to study bioreporter egfp in minute volumes of cell lysates as well as in whole bioreporter bacterial cells, the individual volume of the latter being roughly that of the LIF-CS focal volume. Arsenic, in the aqueous form of arsenite, is our analyte or stressor, an outcome of using the strain Escherichia coli DH5R pPR-arsR-ABS, chosen because it has been well characterized previously and is known to have the suitably reproducible induction response ideal for method development.27 EXPERIMENTAL SECTION Bacterial Strain, Culture Conditions, and Activation. The construction of bioreporter strain E. coli DH5R (pPR-arsR-ABS, (12) Willardson, B. M.; Wilkins, J. F.; Rand, T. A.; Schupp, J. M.; Hill, K. K.; Keim, P.; Jackson, P. J. Appl. Environ. Microbiol. 1998, 64, 1006-1012. (13) Cai, J.; DuBow, M. S. Biodegradation 1997, 8, 105-111. (14) Sticher, P.; Jaspers, M. C. M.; Stemmler, K.; Harms, H.; Zehnder, A. J. B.; van der Meer, J. R. Appl. Environ. Microbiol. 1997, 63, 4053-4060. (15) Rigler, R.; Elson, E. S. Fluorescence Correlation Spectroscopy, Theory and Applications; Springer-Verlag: Berlin, 2001. (16) Eigen, M.; Rigler, R. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5740-5747. (17) Mets, U ¨ .; Rigler, R. J. Fluoresc. 1992, 4, 259-263. (18) Nie, S. M.; Chiu, D. T.; Zare, R. N. Science 1994, 266, 1018-1021. (19) Sauer, M.; Drexhage, K. H.; Zander, C.; Wolfrum, J. Chem. Phys. Lett. 1996, 254, 223-228. (20) Shera, E. B.; Seitzinger, N. K.; Davis, L. M.; Keller, R. A.; Soper, S. A. Chem. Phys. Lett. 1990, 174, 553-557. (21) Trautman, J. K.; Macklin, J. J.; Brus, L. E.; Betzig, E. Nature 1994, 369, 40-42. (22) Weiss, S. Science 1999, 283, 1676-1683. (23) Elson, E. L.; Magde, D. Biopolymers 1974, 13, 1-27. (24) Ehrenber, M.; Rigler, R. Chem. Phys. 1974, 4, 390-401. (25) Hakkila, K.; Maksimow, M.; Karp, M.; Virta, M. Anal. Biochem. 2002, 301, 235-242. (26) Leveau, J. H. J.; Lindow, S. E. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 34463453. (27) Stocker, J.; Balluch, D.; Gsell, M.; Harms, H.; Feliciano, J.; Daunert, S.; Malik, K. A.; Van der Meer, J. R. Environ. Sci. Technol. 2003, 37, 4743-4750.
2684
Analytical Chemistry, Vol. 77, No. 9, May 1, 2005
expressing egfp) used in these experiments is described elsewhere.27 Cells were cultivated by inoculating a single colony, grown on Luria broth (LB) agar, in 5-10 mL of LB for ∼16 h (overnight) at 37 °C and 150 rpm. All LB media contained 50 µg/ mL kanamycin sulfate. The overnight culture was diluted 1:50 in LB and the resulting culture grown under the same conditions to an optical density at 600 nm (OD600) of 0.6. Then, the cells were harvested by centrifugation and resuspended in modified M9 medium (or MM9, MM9 is 100 mL of modified M9 salts solution, 2 mL of 1 M MgSO4, 0.1 mL of 1 M CaCl2 solution, and 10 mL of 20% w/w glucose solution per liter; modified M9 salts solution is 5 g of NaCl, 10 g of NH4Cl, 54.8 g of MOPS free acid, 51.0 g of MOPS sodium salt, 0.59 g of Na2HPO4‚2H2O, and 0.45 g of KH2PO4 in 1 L of water, final solution adjusted to pH 7; MOPS is 3-(Nmorpholino)propanesulfonic acid). Durand et al.28 reported that the growth normalized response of bioreporter luciferase production in response to tributyltin may vary with time, reaching a minimum at maximum growth rate; hence, we use MM9 medium here to prevent the cells from growing as rapidly as in LB medium, thus avoiding both growth-influenced variations in egfp production and the need for large normalization corrections to compensate for the effects of growth on measured response. Quantities of arsenite solutions were added to samples of cells in MM9 to provide the final desired arsenite concentrations and an OD600 of ∼0.6-0.9 (the initial OD600 was constant for all samples within each experiment). Arsenite solutions were prepared fresh from an arsenite standard solution (0.05 M, Merck). The strain we use here is also responsive to other aqueous forms of arsenic (e.g., arsenate). We use arsenite here for convenience, but we note that any eventual analytical protocols utilizing whole-cell biosensors will have to either address possible (chemical) species-specific response characteristics or utilize biocompatible redox buffers to ensure uniformity of chemical speciation. Sample Preparation. Two types of samples were used in LIFCS work: these included lysates and whole cells. Lysates were prepared by taking a 2-mL sample of activated or control (no arsenite) cell suspensions, centrifuging at 15000g for 1 min, removing the supernatant, and then lysing the cells by bead beating with 200 µL of pH 8 tris (10 mM) and 0.2 g of glass beads (0.10-0.11 mm) in a bead beater (Fast Prep FP120, Bio 101 Savant, Vista, CA) for 3 min on the highest setting. After bead beating, samples were centrifuged for 3 min at 15000g, and 5080 µL of lysate was collected. Fresh lysates were analyzed by LIFCS, though we also confirmed that lysates frozen and thawed for subsequent analysis yielded the same results. Whole-cell samples were prepared by direct deposition of 0.5 µL of an OD600 ∼ 0.6 activated cell suspension onto the glass bottom slide of a closedmode POC chamber (H. Saur). After ∼5 min, a layer of cells would form by settling onto the surface of the POC chamber slide. Samples for analysis by epifluorescent microscopy (EFM) were prepared in the same manner, only using a standard microscope slide with a coverslip on top of the cell suspension. Concentrated cell suspensions were made for EFM by centrifuging 100-200 µL of M9 cell suspensions, removing the supernatant, and adding 10-50 µL of pH 8 tris (10 mM). No sample preparation was necessary for analysis by steady-state fluorometry (SSF); activated (28) Durand, M. J.; Thouand, G.; Dancheva-Ivanova, T.; Vachon, P.; DuBow, M. Chemosphere 2003, 52, 103-111.
and control solutions were prepared for direct measurement in 96-well microtiter plates. Sample Analysis. A Confocor spectrometer (Carl Zeiss, Jena, Germany) was used for LIF-CS analysis with Ar ion laser excitation at 488 nm. Fluorescent events were counted by the detector (for 10 s, all lysate samples), and in the case of lysate samples, a correlation function was calculated by the instrument software from the trace of counts as a function of time. Fitting parameters of interest from nonlinear fitting of the correlation function included diffusion time of egfp in the focal volume, average measured counts/molecule egfp, and number of egfp molecules in the laser focal volume. The correspondence between the number of molecules in the focal volume and the egfp concentration can be obtained via calibration with egfp standards; such calibration would be one way of inferring the focal volume, since the focal volume is forthcoming given the concentration (number of molecules/volume) and number of molecules. Calibration curves were constructed using purified egfp (BD Biosciences/ Clontech, supplied as 1 µg/µL egfp in pH 8, 10 mM tris) diluted to final concentrations of 0-100 nM egfp in 10 mM tris at pH 8. Obtaining a fluorescence correlation function can aid in determining whether oligomerization of egfp has occurred. Molecular weight changes associated with such a process are apparent through measured diffusion times. Our studies on lysates yield diffusion times that vary little, ruling out changes in correlation response (molecules/focal volume) due to chemical interactions of this type. Also, comparison of the fluorescence correlation diffusion times for egfp and rhodamine green (given the molecular weight of rhodamine green and that diffusion time is proportional to the cube root of molecular weight) are consistent with the monomeric form of egfp. For whole-cell studies, analysis of the correlation function is considerably more involved; hence, for simplicity a 1-s average detector count rate was obtained. For analysis of lysate samples, the Confocor spectrometer was adjusted so that the beam waist coincided roughly with the middle of the lysate droplets deposited onto a coverslip. In the case of wholecell analysis, the position of the beam waist was adjusted to coincide with a layer of cells just above the POC bottom slide surface and focused accordingly. Although it is possible to focus this instrument with great accuracy and the focal volume is roughly equivalent to the average cell size for the strain used here, the cell volume will not be perfectly coincident with the focal volume, in part because of the great variation in size of and individual shapes of bacteria and in part because of variations in the orientation of each with respect to the microscope slide surface. As this overlap will vary from measurement to measurement, precision will be affected. Our and others’ work illustrates/ predicts that the effect of population dynamics on response will be a greater concern in this respect.29-31 A total of 25 measurements were made at different spot positions, each separated by a lateral distance of at least 10 µm. An Olympus BX-60 with a BX-FLA fluorescence attachment was used for EFM. A minimum of three images of each sample slide were taken with a Spot Insight QE camera (Visitron Systems); exposure times varied from 100 to 500 ms. SSF measurements were made with a BMG Fluostar Galaxy microplate (29) Miller, W. G.; Brandl, M. T.; Quinones, B.; Lindow, S. E. Appl. Environ. Microbiol. 2001, 67, 1308-1317. (30) Stiner, L.; Halverson, L. J. Appl. Environ. Microbiol. 2002, 68, 1962-1971.
measurement system with a 56-s fluorescence intensity measurement cycle (10 flashes, 0.2-s delay, gain 10, λex ) 480 nm, λem ) 520 nm) programmed at each sampling time followed immediately by measurement of OD600 (gain 34). Raw Data Treatment. No treatment of analytical data was required for LIF-CS analyses of lysates, the lysates themselves representing an average egfp response for a large population (∼109 cells). Three replicate measurements were made for each sample. Analyses of whole cells involved 25 measurements/sample, and results are reported as an average of the 25th-75th percentile response (i.e., to eliminate outliers). EFM images were analyzed by a Metamorph software routine that involved image optimization for counting of fluorescent spots, followed by collection of statistics on each spot (size, shape factor, average gray value or AGV). To measure cellular AGVs, objects were filtered according to expected size and shape factor. Then, to “boost signal”, the 75th-90th percentile background-subtracted average of all cellular AGVs per image was recorded. Three “replicate” analyses, consisting of three images, were made for each sample; the average number of bacteria in each image averaged ∼500. Per LIF-CS measurements on lysates, SSF measurements also represent an average population response; eight replicate measurements were performed per SSF sample with no added treatment of analytical data. LIF-CS, EFM, and SSF each involve different constraints in method optimization and, hence, the different number of replicate measurements and different approaches to data analysis. For data expressing egfp evolution as a function of time, we applied a t growth correction factor of ODt)0 600 /OD600, where OD600 is the optical density of the bacterial culture at 600 nm and t is the elapsed induction time. Data expressing egfp as a function of concentration are cotemporaneous and thus not corrected for growth; hence, the ordinate response values may be higher, depending upon the induction time. Given variability of biological systems, there is some variability in response for independent experiments as well. Calculations. To assess the LIF-CS lysate method described here, we calculated figures of merit (FOM) for arsenite analyses from all methods. FOM include analyte MDL, average relative standard deviation (RSD) for replicate measurements, linear dynamic range (LDR), sc (the uncertainty associated with an unknown determination using a calibration curve, in this case at a point halfway to the upper limit of the LDR on a calibration curve), S (calibration sensitivity), and R2 (correlation coefficient of calibration curve). For brevity, selected FOM are reported in Table 1 of the article, with the remainder being in Supporting Information Table 1. MDLs were also calculated for egfp detection. Where possible, we determine MDLs in the standard manner, e.g., 3sby, as recommended by Skoog et al.32 and other standard texts (where sby is the standard deviation of measurements on a blank). When measuring the reporter response to arsenite in whole cells and lysates, this works well as the baseline reporter production of egfp is nonzero. However, for a calibration curve using egfp standards, a zero egfp concentration blank is used, resulting in poor fluorescence autocorrelation. Thus, the MDLLIF-CS (for egfp in buffer) was determined as 2sby, where sby egfp (31) Wells, M.; Go ¨sch, M.; Harms, H.; van der Meer, J. R. Microchim. Acta, submitted. (32) Skoog, D. A.; Holler, F. J.; Nieman, T. A. Principles of Instrumental Analysis, 5th ed.; Saunders: Philadelphia, 1998.
Analytical Chemistry, Vol. 77, No. 9, May 1, 2005
2685
Table 1. Figures of Merit Associated with the Linear Response of PPR-arsR-ABS to Arsenite Exposure as a Function of Concentration at Different Induction Timesa MDLb (nM arsenite)
LIF-CSe EFMf SSF
average RSDc (%)
sensitivityd
0.5 h
1h
2h
3h
4h
0.5 h
1h
2h
3h
4h
0.5 h
1h
2h
3h
4h
270 207 250
69 35 62
9.5 97 67
4.5 40 22
4.3 37 30
3.5 32 1.9
3.7 15 2.1
2.6 30 2.9
3.1 22 4.5
1.7 20 5.2
100 100 100
370 160 510
730 180 1000
1400 680 1900
2200 650 3000
a Note: the LIF-CS method pertains to lysates and is a “bulk” measurement. The EFM method involves measuring individual whole cells, and the SSF measurement is a bulk measurement, but also using whole cells. See Supporting Information for additional FOM. b Method detection limit, concentration equivalent of 3sbx response, where sbx is the standard deviation of the “blank” (no arsenite). c Relative standard deviation, mean of three replicate measurements (LIF-CS), mean of three image AGVs (EFM), mean of eight replicate measurements (SSF), all for a single sample (i.e., intraexperimental, single arsenite concentration). d Percent increase from that at t ) 0.5 h; sensitivity is much higher for SSF than for LIF-CS, but induction time-dependent increases in blank uncertainty result in higher MDLs for SSF than for LIF-CS. e LIF-CS lysate results agree well with FOM for the whole-cell data in Figure 2 for which the MDL is ∼120 nM arsenite (compare to projected 40 nM for lysates at the same induction time), and the RSD of pooled measurements is ∼9% (expected to be higher as it represents pooled measurements of different cells as opposed to repeated measurements of a single drop). f The results for EFM here agree roughly with those of Stocker et al.27 for pPR-arsR-ABS in terms of sensitivity, measurement RSD, and MDL.
Figure 1. Response of strain pPR-arsR-ABS to arsenite exposure as a function of (A) time and (B) concentration, as measured by two independent LIF-CS experiments on cell lysates. The number of fluorophores per focal volume were obtained from experimentally determined fluorescence autocorrelation curves. Results in panel A are corrected for growth. For both panels, each point represents the average of three analyses on a single sample. The 0 µM arsenite response for panel B is 31 ( 6 molecules/focal volume (not shown due to logarithmic plotting scale). Note: in many cases, the lysate egfp concentrations are too high to measure by the LIF-CS single-molecule technique; thus ordinate axis values represent dilution-corrected values.
is the standard ordinate error of a calibration intercept, i.e., the zero from a plot of LIF-CS molecules egfp/focal volume as a function of egfp concentration. The factor of 2 is used in this case since intercept uncertainty resulting from several regressors is more conservative than the standard deviation of a blank measurement (note: some authors use a factor of 2 criterion as a matter of course even for standard deviation of a blank27). The LIF-CS arsenite MDLs decrease roughly exponentially with increasing induction time (see Supporting Information Figure 1). From such a trend, we estimate the minimum induction time necessary to achieve arsenite MDLs of interest, as described in the Results and Discussion (note: for reasons discussed below, we find that, at present, measurements at induction times of less than 30 min are not routinely possible). Measurements on lysates enable the characterization of matrix effects via spike recoveries. Spike recoveries in this study ranged from 70 to 600%. Recoveries greater than 100% were exclusively limited to low egfp/low induction time samples (t < 0.5 h), whereas recoveries below 100% occurred only at very high egfp concentrations, neither observation being unusual for matrix interferences in fluorescence work. For details of other calcula2686
Analytical Chemistry, Vol. 77, No. 9, May 1, 2005
tions (rate of egfp production/nmol analyte in solution, maturationlimited minimum induction time, pPR-arsR-ABS intracellular baseline egfp concentration, quantum yield/fluorophore extinction coefficient corrections for comparison of gfp literature results to present data), see the Supporting Information. RESULTS AND DISCUSSION Strain pPR-arsR-ABS was exposed to different aqueous arsenite concentrations and the time-dependent egfp response measured on lysates (Figure 1A); similarly, the concentration-dependent response was measured at different induction times (Figure 1B). In Figure 1A, bioreporter background egfp expression, i.e., egfp present at 0 µM of arsenite, is well above the ∼2.6 molecules/ focal volume MDLLIF-CS ; the response increases monotonically egfp over the first 4 h and exhibits scatter at t < 0.5 h. Visual inspection and linear regression of the t g 1 h data in Figure 1A suggests a minimum induction time of ∼1 h to adequately distinguish basal egfp expression from arsenic-induced signal (all concentrations), but exponential fits to the t ) 0-4 h data in Figure 1A indicate minimum induction times of 50, 13, and 9 min for 0.1, 1.0, and 10 µM arsenite, respectively (see Supporting Information for further
discussion of fitting models). These numbers do not address the issue of scatter in the t e 0.5 h data, and if fitting is confined to this shorter response range, a different result is obtained. At t ) 0.5 h, the response in Figure 1B for 10 µM arsenite is more than 300 times the corresponding 0 µM response; i.e., response for the blank versus 10 µM arsenite is readily distinguished statistically. We find that the minimum induction time to distinguish basal expression from arsenic-induced signal is always 0.5 h or less, but is subject to the variability that can occur with measurements on biological systems. Minimum induction time for detection of an increase in bioreporter signal could be limited by measurement uncertainty associated with variations in background expression of egfp by pPR-arsR-ABS. However, in the course of this work, we observed negligible variations in intraexperiment replicate measurements of the background signal. Another possible constraint on the minimum induction time for gfp/egfp reporters is fluorophore maturation. Using literature data (first-order maturation kinetics and a maturation constant of 1.5 h-1 33), we predict34 a maturationlimited necessary minimum induction time of ∼7 min, therefore an operative but not limiting process here. In fact, unlike chemical methods of analysis, egfp background may be beneficial; firstorder maturation kinetics dictates that the maturation rate Rm ) kCegfp (where k is a rate constant and Cegfp is the intracellular egfp concentration), indicating that the maturation of de novo induced egfp is faster with a larger background Cegfp. Concerns about limitations as such imposed by first-order maturation kinetics was one reason we chose not to perform these initial experiments with an ultralow background reporter strain despite the LIF-CS inherent single-molecule detection capability; our experience to date with such low background reporters suggests that they may be intrinsically more challenging to induce reproducibly at low activation times. This is a subject of ongoing research in our laboratories, and beyond the scope of the present article. We find that the main limiting factor governing the minimum induction time in Figure 1 is matrix interference with the lower levels of egfp present at low induction times. A quantitative indicator of matrix interference is spike recovery, 100% recovery indicating no interference; for this study, purified egfp in buffer was used as the spiking solution and lysate samples were spiked with known quantities of egfp. Spike recoveries were typically ∼100% for lysate samples measured at t > 0.5 h induction, indicating no matrix interference. Spike recoveries for zero arsenite concentration lysate samples were, on average, 600% (indicating substantial matrix interference) and nonlinearly approached 100% with increasing induction time, increasing Cegfp, or both. Thus matrix-induced analytical bias leads to the scatter in the Figure 1A data for t < 0.5 h and is the main factor limiting the minimum necessary induction time. Various mechanisms could explain high recoveries at low egfp lysate concentrations, but the important point is that correcting for these spike recoveries implies that the actual egfp lysate background is ∼6 molecules/ focal volume and that the true linear range response sensitivity (hence, bioreporter sensitivity to arsenite) is greater than one would suppose from the present data or that of Stocker et al.27 (33) Heim, R.; Cubitt, A. B.; Tsien, R. Y. Nature 1995, 373, 663-664. (34) See Supporting Information.
We estimate an average gross lysate egfp production of 10, 60, and 150 nM/h for 0.1, 1.0, and 10 µM arsenite, respectively, based on both time-dependent and concentration-dependent induction experiments (the interested reader is referred to the Supporting Information for details of these calculations34). This amounts to about 0.01, 0.006, and 0.001 nmol/h egfp produced per nanomole of arsenite in solutions at 0.1, 1, and 10 µM arsenite, respectively.34 This apparent “stoichiometry” suggests that a disproportionate amount of stressor would be required to produce a detectable response but may be misleading. Intracellular arsenite concentrations may actually be low because of the interplay of limitations in uptake35 and concomitant facilitated cellular efflux.36 The diminution in bioreporter response rate with increasing arsenite concentration that we report here has been observed previously,27 though not quantitatively in terms of egfp. Arsenic toxicity is a logical explanation for this observation and the leveling/diminution of the rate of egfp production (Figure 1B) for arsenite concentrations above ∼3 µM. The possibility of false negatives arising from even higher arsenite concentrations (i.e., 100% effective cell death) could be addressed via analysis of dilution series. For some applications, this would never become a practical issue; for instance, with respect to environmental applications, the highest arsenite concentration exhibited in Figure 1B (100 µM) is substantially above the highest reported well water concentrations in Bangladesh.37,38 Though LIF-CS is ideal for in vivo analysis of cellular processes,15 there are no reports as yet, to our knowledge, of such studies on bioreporters. Analysis of whole bioreporter cells has advantages over analysis of lysates, e.g., shorter analysis time, lower sample volume, higher throughput, reduction of errors attendant upon reduced method complexity, and in situ measurements at the cellular level for understanding the dynamics of organismal response to toxic stress. For the pPR-arsR-ABS cell line, the average cell volume is near that of the LIF-CS focal volume; this substantially decreases measurable fluctuations in the signal and constrains the fluorophores in a manner that could increase photobleaching, quenching, or both. These factors complicate deconvolution of experimental autocorrelation data however, it is still possible to measure an average count rate for fluorescent events within the small focal volume, and the average count rate suffices to monitor pPR-arsR-ABS response to arsenic. Figure 2 shows such a spectroscopic response for whole cells as a function of arsenic concentration. Also in Figure 2, for comparison, is the lysate response curve, which agrees quite well with the whole-cell results, the only difference being that the wholecell response drops off more rapidly than do the lysate response trends. The more rapid diminution in whole-cell response at high arsenite concentrations probably results from inherent differences in the measurements between LIF-CS of lysates and spectroscopic determinations on whole cells. The sampling of lysates ensures that a “freeze-frame” picture of the egfp in the cells is taken; i.e., (35) van der Meer, J. R.; Tropel, D.; Jaspers, M. Environ. Microbiol. 2004, 6, 1005-1021. (36) Dey, S.; Dou, D.; Rosen, B. P. J. Biol. Chem. 1994, 269, 25442-25446. (37) Hug, S. J.; Canonica, L.; Wegelin, M.; Gechter, D.; Von Gunten, U. Environ. Sci. Technol. 2001, 35, 2114-2121. (38) 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.
Analytical Chemistry, Vol. 77, No. 9, May 1, 2005
2687
Figure 2. Total LIF-CS detector counts (squares) measured from fluorescence of activated whole pPR-arsR-ABS cells as a function of arsenite concentration (induction time is 1.5 h). Lysate response (circles) is shown for comparison (data are an average of t ) 1 h and t ) 2 h traces from Figure 1B); molecules/focal volume obtained from LIF-CS fluorescence autocorrelation curve. Selected EFM images shown for comparison.
because cells are lysed and then the abiotic lysate immediately separated from cell components, a sample with static egfp content is acquired. Alternately, since the measurement on whole cells utilizes cells that are living, there is a small amount of uncertainty associated with the sampling time (manifest more clearly at high arsenite concentrations where cells are experiencing inhibition). We are currently developing automation protocols to address this issue in whole-cell analysis. To assess the LIF-CS detection scheme, we list selected FOM in Table 1 for the present lysate protocol along with those for two additional protocols employing conventional detection schemes (EFM and SSF, notesadditional FOM are available in the Supporting Information). Scott et al.39 reported a 1 order of magnitude decrease in MDLs for bioreporter detection of arsenite and antimonite from 0.5- and 2-h induction followed by another order of magnitude decrease between 2- and 17-h induction. Similarly, our bioreporter LIF-CS arsenite MDLs decrease exponentially with induction time. The minimum estimated induction times to detect, for instance, the U.S. Environmental Protection Agency (50 µg/L) and World Health Organization (10 µg/L) limits on arsenic in drinking water using the lysate protocol are
