Simultaneous detection of analytes based on genetically engineered whole cell sensing systems

Analytica Chimica Acta 444 (2001) 251–260

Simultaneous detection of analytes based on geneticallyengineered whole cell sensing systems

Suresh Shrestha, Ranjit S. Shetty, Sridhar Ramanathan, Sylvia Daunert∗Departments of Chemistry and Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40506-0055, USA

Received 15 February 2001; received in revised form 8 June 2001; accepted 20 June 2001

Abstract

Sensing systems for the simultaneous detection of analytes utilizing whole cells provide efficient ways to develop analyticalassay systems with reduced cost. In this work, a whole cell-based sensing system was developed for the simultaneous detectionof two model analytes,�-lactose andl-arabinose, using genetically-designed bacteria. Two variants of the green fluorescentprotein (GFP), BFP2 and GFPuv, were used as the reporter protein for the detection of each analyte. The corresponding reportergenes were introduced into the bacteria in such a way that they can be co-expressed along with the other genes induced bythe respective sugar. Each of these fluorescent proteins is expressed only in the presence of their respective analyte and theirfluorescence emission can be monitored using the intact whole cell samples. By exciting the cells at 380 nm, emission forBFP2 and GFPuv can be collected at 440 and 509 nm, respectively. The fluorescence emission thus obtained can be correlatedwith the amount of sugars present in the sample. Calibration curves for�-lactose andl-arabinose were generated. It wasobserved that this system shows no significant response to other closely related sugars thus providing high selectivity for�-lactose andl-arabinose detection. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Detection of analytes; Genetically-designed bacteria; Model analytes

1. Introduction

In recent years, multianalyte assay development hasreceived considerable attention as it allows simultane-ous detection of multiple analytes with reduced time,cost, and sample size when compared to traditionalindividual analyte testing [1,2]. The simultaneous de-tection of various analytes is typically achieved byusing labels that produce distinguishable signals. Thishas been demonstrated using labels such as, fluores-cent molecules and proteins [3–5], radioactive labels[6,7], enzymes [8,9], lanthanides [10], and colored

∗ Corresponding author. Tel.:+1-859-257-7060;fax: +1-859-323-1069.E-mail address: [email protected] (S. Daunert).

latex particles [11]. Most of these labels have been em-ployed in the development of multianalyte immuno-assays using antibodies or antigens conjugated todifferent labels [12]. In this study, we have developeda sensing system for the simultaneous detection oftwo analytes in whole cells using fluorescent reporterproteins. For that, we selected two sugars,�-lactoseandl-arabinose, as model analytes.

Cell-based sensing systems using reporter proteinshave been developed for diverse chemicals such as,organics [13–16], oxoanions [17–19], metals [20–22],and a pentose sugar [23]. Whole cell-based sensingsystems have several distinct advantages over sensingsystems that employ isolated biological components[24,25]. For example, intact cells employed in thesesensing systems are more tolerant to assay conditions

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0003-2670(01)01214-4

252 S. Shrestha et al. / Analytica Chimica Acta 444 (2001) 251–260

and can give information about the effect of the an-alyte on the physiological state of the cell. Thesesystems do not require isolation of biological compo-nents, thus reducing the overall cost of the assay. Theavailability of various reporter genes has enabled re-searchers to develop whole cell sensing systems withbioluminescence, chemiluminescence, or fluorescencedetection [17,18,23].

In the present study, we have employed two vari-ants of the green fluorescent protein (GFP), GFPuvand BFP2, to develop a bacterial cell-based sensingsystem for two different sugar analytes. The devel-opment of detection methods for sugar analytes hastraditionally been difficult as they lack chromophoricunits in their structures. The structures of sugars arevery similar and therefore, the electrochemical detec-tion methods developed so far are not very specific[26]. To overcome these problems, sugars usuallyneed to be derivatized before employing an analyticaldetection method [27,28]. In this study, we take ad-vantage of certain proteins present in cells that havespecific binding affinities for sugars to avoid deriva-tization steps and be able to develop direct sensingmethod for sugar(s). For the detection, we have em-ployed fluorescent reporter proteins as they allowdirect measurement of the signal through whole cells.

GFP is one of the best reporters available as it canbe used as a non-invasive indicator of gene expressionwithout the need to add any substrates in order togenerate a signal. The introduction of various mutants

Fig. 1. (A) Plasmids pSD909 and pBAD-GFPuv and (B) picture of the recombinant bacteria with expressed BFP2 and GFPuv induced by�-lactose andl-arabinose, respectively.

of GFP with altered spectral properties has permittedthe simultaneous imaging of different proteins in cells[29,30]. GFPuv has been optimized for excitation byUV light and for higher expression in bacterial cells.Three amino acid substitutions, Phe 99 to Ser, Met153 to Thr, and Val 163 to Ala, on the wild-type GFPgave rise to GFPuv [31]. Similarly, the BFP2 variantwas created from four mutations, Phe 64 to Leu, Ser65 to Thr, Tyr 66 to His, and Tyr 145 to Phe on thewild-type GFP [32]. These mutants show increasedefficiency in chromophore formation due to improvedprotein folding and enhanced solubility, and hence,produce stronger fluorescence [31–33].

�-Lactose andl-arabinose are taken up and metab-olized by bacteria through thelac and ara operons,respectively. These sugars induce the expressionof genes in their respective operons, which arenecessary for their transport into the cell and fortheir metabolism. The plasmids, pBAD-GFPuv andpSD909, were employed in this study for the devel-opment of a whole cell dual analyte sensing system(Fig. 1). The plasmid pBAD-GFPuv consists of thegene for AraC, which acts as an activator of thearaoperon, and thegfpuv gene under the control of thePBAD promoter. The activator protein AraC bindsl-arabinose and regulates the expression of GFPuv.The plasmid pSD909 contains the gene,bfp2, underthe control of thetac promoter, and it is regulated byLacI, the repressor protein involved in the negativeregulation of thelac operon. In the presence of lactose,

S. Shrestha et al. / Analytica Chimica Acta 444 (2001) 251–260 253

repression of thelac operon by LacI is reversed lead-ing to the expression of BFP2. The cells containingthe plasmids pBAD-GFPuv and pSD909 were mixedand the expression of reporter proteins was inducedby adding the mixture of�-lactose andl-arabinose.The reporter proteins expressed can be characterizedby monitoring their fluorescence emission using theintact whole cell samples. These reporter proteins canbe both excited at 380 nm and their fluorescence emis-sion can be collected at 440 and 509 nm for BFP2 andGFPuv, respectively. The amount of fluorescence in-tensity thus obtained can be correlated to the amount ofthe individual sugars in the sample. Calibration curvesfor both sugars were constructed and the selectivity ofthis system was determined.

2. Experimental

2.1. Reagents

Plasmid pFLAG-MAC was obtained from Sigma(St. Louis, MO). Plasmids pBAD-GFPuv and pBFP2were purchased from Clonetech (Palo Alto, CA).E.coli strains BL21(DE3) and JM109 were obtainedfrom Stratagene (La Jolla, CA). Sodium phosphate,�-lactose,d- andl-arabinose,d-fructose,d-mannose,d- andl-xylose,d-ribose,d- andl-lyxose,d-glucose,maltose, and all other reagents were from Sigma(St. Louis, MO). All the molecular biology reagentswere purchased from Gibco-BRL (Gaitherburg, MD).The primers used for the polymerase chain reac-tion (PCR) were obtained from Operon Technologies(Alameda, CA). Solutions were prepared using deion-ized (Milli-Q Water Purification System, Millipore,Bedford, MA) distilled water. All chemicals werereagent grade or better and were used as received.

2.2. Apparatus

PCR was performed on a Perkin-Elmer Gene AmpPCR System 2400 (Norwalk, CT). Bacterial cells weregrown in an orbital shaker (Forma Scientific, Mari-etta, OH). Fluorescence measurements were taken ina SPEX Fluorolog-2 spectrofluorometer (SPEX In-dustries, Edison, NJ) using disposable methacrylatecuvettes purchased from Fisher Scientific (Pittsburg,PA). Fluorescence intensities reported are the average

of a minimum of three replicates and have been cor-rected for the contribution of the blank. All molecu-lar biology procedures were performed using standardprotocols [34].

2.3. Construction of pSD909

To obtain the gene encoding for BFP2, PCR wasperformed using the plasmid pBFP2 as a template. Thefollowing oligonucleotides were used for the PCR:(1) TCTTCTTCTTCTTCTAAGCTTATGAGTAAA-GGAGAAGAA; (2) TCAGTTGGAATTCTACGAA-TGCTATTTGTATAGTTCATC. The underlined basesencode for the restriction enzyme cleavage sites,HindIII in (1) and EcoR I in (2). The DNA construct thusobtained and the plasmid pFLAG-MAC were digestedwith Hind III and EcoR I. The digested products wereligated to obtain pSD909 (see Fig. 1). AnE. colistrain BL21(DE3) was transformed with pSD909.A single colony of the transformed bacteria was se-lected and grown overnight. A mini-prep plasmidwas prepared and the presence ofbfp2 was confirmedby DNA sequencing at the Macromolecular Center(University of Kentucky). Similarly,E. coli strainJM109 was transformed with plasmid pBAD-GFPuv.For transforming plasmid pSD909, theE. coli strainBL21(DE3) was employed instead of strain JM109,because JM109 lacks thelacZ gene, which is essentialfor the functioning of thelac operon.

2.4. Fluorescence characterizationof mixture of cells

Bacteria containing plasmid pBAD-GFPuv orpSD909 were scraped from the glycerol stock andgrown in agar plate. A colony was picked from eachplate and grown separately in 4.0 ml of LB mediumcontaining 100�g/ml of ampicillin at 37◦C until itreached an optical density value at 600 nm of 0.6(OD600 of 0.1 = 1× 108 cells/ml) [35]. A 1.0 ml vol-ume of culture containing cells with pBAD-GFPuvwas mixed with 1.0 ml of cells containing pSD909.�-Lactose andl-arabinose were added to the mixtureof cells and the final concentration of each was ad-justed to 1× 10−3 M. The cells were allowed to growfor an additional 3 h. After this time period, the cellmixture was centrifuged at 10,000× g for 5 min andthe supernatant was discarded. The bacterial pellet


was washed twice with 10 mM phosphate buffer, pH7.5 (phosphate buffer). In each wash step, the pelletwas resuspended in 1 ml of the buffer. The bacterialsuspension was finally diluted to a volume of 6.0 mlwith the phosphate buffer. Fluorescence excitationand emission scans were obtained using 1.5 ml ofdiluted cells.

2.5. Induction-time study

Cells containing plasmid pBAD-GFPuv or pSD909were grown in 200 ml of LB medium containing100�g/ml of ampicillin at 37◦C until an absorbancevalue at 600 nm of 0.6 was obtained, as describedpreviously [35]. A 1.0 ml volume of each of the twodifferent cell cultures were mixed together and thetwo sugars,�-lactose andl-arabinose, were addedand the final concentration of each was adjusted to1 × 10−3 M. The cells were then allowed to grow forvarious time periods using a fresh mixture of cells foreach time period. The cells were harvested at the endof each time period by centrifuging at 10,000× g for5 min and the supernatant was discarded. The pelletwas resuspended and diluted to a final volume of6.0 ml with phosphate buffer after washing the pelletas described previously. The fluorescence intensity ofthe cells was measured using the sample volume of1.5 ml in a cuvette by exciting the cells at 380 nm.The BFP2 and the GFPuv emissions were collectedat 443 and 506 nm, respectively. Control studies werealso performed in the same way as described abovewithout any addition of sugars.

2.6. Calibration study for β-lactoseand l-arabinose

Standard solutions of different concentrations of�-lactose andl-arabinose were prepared by serialdilution. A 1.0 ml each of freshly grown cells con-taining plasmid pBAD-GFPuv and cells with pSD909both with an absorbance value at 600 nm of 0.6 weremixed together. Various concentrations of�-lactoseand l-arabinose were added to the mixed cells. Afresh mixture of cells was used for each of the con-centration of sugars chosen. No sugars were added tothe control sample. The cells were incubated at 37◦Cfor an additional 3 h. Then, the fluorescence inten-sity was measured using a sample volume of 1.5 ml

after following the washing and dilution proceduredescribed above.

2.7. Selectivity studies

The selectivity of the dual-analyte sensing systemwas evaluated using various sugars. The response ofd-arabinose,d-fructose,d-mannose,d- andl-xylose,d-ribose,d- andl-lyxose,d-glucose, and maltose wasstudied. For that, a final concentration of 1×10−3 M ofthe sugars was added to the mixture of cells preparedas described previously, and the fluorescence intensitywas measured at 443 and 506 nm for BFP2 and GFPuv,respectively, after excitation at 380 nm.

3. Results and discussion

Green fluorescent protein isolated from the jelly-fish Aequorea victoria has become a popular reporterprotein and an in vivo cell marker since the cloning ofits cDNA [30]. Some of the advantages of GFP whichinclude a high fluorescence quantum yield (∼0.85),no substrate requirement, stability to pH, temper-ature, and photobleaching have made it a uniquereporter [36,37]. GFP has been employed in diverseapplications such as monitoring gene expression, de-velopment of binding assays, and as a reporter proteinin cell-based assays and cellular imaging [36]. Theintroduction of various mutants of GFP has furtherdiversified its applications in cell biology and in an-alytical assay systems. These mutants with differingspectral properties have enhanced fluorescence andincreased stability compared to the wild-type GFP. Inthat respect, the aim of this study was to develop adual analyte cell-based sensing system taking advan-tage of distinguishable emission characteristics of themutants of GFP. Specifically, a sensing system wasdeveloped for two sugar analytes, namely,�-lactoseandl-arabinose. In bacteria, these sugars act as alter-nate sources of energy for the cell in the absence ofglucose and regulate the expression of the genes ofthe operon involved in their uptake and metabolism.The sensing system described here utilizes engineeredbacteria that contain a plasmid with the genes ofthe protein that regulate sugar and that code for thereporter protein. The reporter proteins are expressedin the presence of the sugar analytes that bind to the


Fig. 2. Schematic of the regulation and expression of BFP2 in the presence of�-lactose in pSD909. O/P represents the operator/promoterregion.

regulatory protein, allowing then for transcription andtranslation of the reporter protein to occur.

Lactose is involved in the negative regulation ofthe lac operon (Fig. 2). Thelac operon consists ofthree structural genes,lacZ, lacY, and lacA. The en-zyme �-galactosidase, responsible for lactose break-down, is encoded bylacZ. In the absence of lactose,the repressor protein LacI, expressed independentlyfrom lac operon, binds to the operator/promoter regionof the lac operon and prevents transcription. Lactosewhen present, enters the cell utilizing basal amountof permease, encoded bylacY, and is converted to al-lolactose. Allolactose binds to the LacI protein andthen, the LacI-allolactose complex is released fromthe operator/promoter region allowing for transcrip-tion of the genes in thelac operon to occur [38]. Inour studies, we constructed plasmid pSD909 by in-troducing thebfp2 gene into the pFLAG-MAC vec-tor (Fig. 1). The plasmid pFLAG-MAC consists of atac promoter under the regulation of LacI. The genethat encodes for the blue fluorescent protein, BFP2,was fused downstream from thetac promoter.E. colistrain BL21(DE3) was then transformed with the plas-mid pSD909. These cells harboring pSD909 are beinginduced by lactose, which in turn allows for the tran-scription and translation ofbfp2 gene to commence.

The second sugar system that we employed in ourstudies isl-arabinose, which is regulated by thearaoperon. The regulatory protein AraC acts as a positiveregulator of theara operon (Fig. 3). In the presenceof l-arabinose, it forms the AraC–l-arabinose com-plex and binds to the activator region of the operon.This initiates the transcription of the genes of theoperon [23]. The plasmid that we used in our work ispBAD-GFPuv, which consists of thegfpuv gene en-coding for GFPuv under the control of thePBAD pro-moter (Fig. 1). The expression of GFPuv is regulatedby thearaC present in the plasmid. Thus, the amountof GFPuv expressed can be correlated to the amountof l-arabinose present in the sample. A whole cellsensing system forl-arabinose has been previouslydeveloped in our laboratory using cells harboring thisplasmid [23]. In the present study, this scheme iscoupled to the lactose system for the simultaneousdetection ofl-arabinose and�-lactose.

A study was performed to correlate the expressionof BFP2 with the amount of�-lactose to which cellscontaining pSD909 were exposed. To perform thisstudy, bacteria harboring plasmid pSD909 were in-duced with�-lactose to a final concentration of 1×10−3 M for 3 h. The fluorescence of expressed BFP2was monitored exciting the intact cells at 380 nm and


Fig. 3. Schematic of the regulation and expression of GFPuv in the presence ofl-arabinose in pBAD-GFPuv.

an emission scan was collected. The control study wasalso performed in the same manner but without anyaddition of �-lactose. The fluorescence spectra ob-tained showed that the reporter protein BFP2 was ex-pressed only in the presence of�-lactose. The controlstudy showed no significant expression of BFP2. Thisdemonstrated that expression of BFP2 is dependenton the presence of�-lactose, and that it can be em-ployed in the development of a detection system for�-lactose. A similar study was performed to correlateGFPuv expression tol-arabinose. Likewise a correla-tion between the concentration ofl-arabinose and theamount of GFPuv produced by the cells was observed.

The first experiment in the development of thedual-analyte sensing system was to characterize thefluorescence of expressed GFPuv and BFP2 in thepresence ofl-arabinose and�-lactose, respectively.For this study, the cells containing pBAD-GFPuvand pSD909 were mixed together after growing themindividually. A final concentration of 1× 10−3 M ofl-arabinose and 1× 10−3 M of �-lactose was addedto the cells and the cells were grown for an addi-tional 3 h. After this, the cells were centrifuged andresuspended in phosphate buffer. The excitation andemission scans were obtained from the resuspendedwhole cell samples (Fig. 4). From the excitation

scan, a wavelength of 380 nm was selected as theexcitation wavelength for further studies. Emissionwavelengths of 443 nm for BFP2 and of 506 nm forGFPuv, respectively, were selected for the emissionscans. These wavelengths are characteristics of therespective proteins. There are no overlaps betweenthe emission spectra of BFP2 and GFPuv at the wave-length selected, which minimizes possible spectralinterferences.

Induction time studies were performed with themixture of cells containing the plasmids pBAD-GFPuvand pSD909. Cells were induced withl-arabinoseand�-lactose for various time periods. A typical in-duction time curve is shown in Fig. 5. No significantincrease in fluorescence was observed after 3 h ofinduction. Therefore, an induction time of 3 h wasselected for generating calibration curves. If desired,the induction time can be reduced to 1 h or less asseen from Fig. 5. While these shorter induction timeswould result in faster assay times, the resulting systemwould have poorer detection limits [23].

The next step in the study was to generate calibra-tion curves for�-lactose andl-arabinose using themixture of cells. Various concentrations of�-lactoseandl-arabinose were introduced to the cells for 3 h.The fluorescence emitted by the mixture of whole


Fig. 4. Excitation scan (left) and emission scan (right) of mixture of cells harboring plasmids pBAD-GFPuv and pSD909.

cells induced with varying concentration of sugars wasmeasured. The calibration curves generated are shownin Fig. 6. Calibration curves obtained show typicalsigmoidal shape and saturation in fluorescence inten-sity was observed at a concentration of 1× 10−2 Mfor both the sugars. The increasing amounts of sugarsgave rise to an increase in the fluorescence intensityfrom both reporter proteins. At lower concentrationsof l-arabinose or�-lactose, the fluorescence intensityfrom the respective reporter protein is lower as the

Fig. 5. Induction time studies of mixture of cells induced at a concentration of 1× 10−3 M of �-lactose orl-arabinose for various timeperiods: (�) cells induced withl-arabinose; (�) cells induced with�-lactose. Data are mean± S.D. (n = 3).

transcription of the genes controlled by the regulatoryproteins, AraC and LacI, is restricted.l-Arabinoseinduction of GFPuv resulted in a maximum of 181%increase in fluorescence intensity, while�-lactoseinduction of BFP2 showed a maximum of a 147% in-crease in fluorescence. A linear range of three orders ofmagnitude extending from a concentration of the sug-ars of 1×10−3 to 1×10−6 M was obtained (the corre-lation coefficients were 0.98 and 0.94 forl-arabinoseand �-lactose, respectively). The response of the


Fig. 6. Calibration curves forl-arabinose (�) and �-lactose (�). The mixture was induced with differing concentrations of sugars for3 h, before measuring fluorescence of BFP2 at 443 nm and of GFPuv at 506 nm. Data are mean± S.D. (n = 3).

sensing system was tested over several days and itshowed no significant variation. Day-to-day relativestandard deviation obtained using triplicate sampleswere <10% for lactose sensing and<12% for ara-binose sensing. The detection limit forl-arabinosewas found to be 5× 10−7 M and a detection limit of1 × 10−6 M was obtained for�-lactose (the detec-tion limit is defined as the sugar concentration thatcorresponds to a S/N= 3).

The selectivity of the whole cell sensing systemwas tested against structurally similar sugars such asd-arabinose,d-fructose,d-mannose,d- andl-xylose,d-ribose, d- and l-lyxose, d-glucose, and maltose.None of the above sugars at concentrations as highas 1× 10−3 M showed any significant interference(Fig. 7). The system is very selective for�-lactose andl-arabinose. Studies were also performed inducingthe mixture of cells only with�-lactose and only withl-arabinose to investigate any possible cross-inductionof the sugars to the two different plasmid systems.From this study, it was found thatl-arabinose doesnot induce the expression of BFP2, and that�-lactoseshows some induction of GFPuv. Nevertheless, thisinduction of GFPuv by�-lactose is small (43% in-crease in signal) compared to the induction causedby l-arabinose (230% increase in the signal) andtherefore, it should have little effect on the assay per-formance. The percent increase in BFP2 fluorescence

intensity was found to be higher when the cells wereinduced with the mixture of sugars than when in-duced with�-lactose alone. This maybe due to thepresence of a synergistic effect on the bacteria in thepresence of the mixture of sugars, asl-arabinose isa better carbon source than�-lactose. This may haveresulted in a high amount of BFP2 expressed whenthe cells were induced with mixture of sugars. Whenonly l-arabinose was added to the cells, the percentincrease in the fluorescence intensity of GFPuv washigher compared to the intensity obtained from addi-tion of the mixture of sugars. This can be explained onthe basis that when the mixture of sugars were addedboth the proteins were expressed and the excitationlight was distributed between the two fluorescent pro-teins. When cells were induced just withl-arabinose,only GFPuv was expressed, and the excitation lightwas absorbed by only this protein, and hence, higherintensity of fluorescence from GFPuv was observed.

In conclusion, we have demonstrated that wholecell-based sensing systems can be developed for the si-multaneous detection of two analytes using variants ofGFP as the signal-generating reporter proteins. A sens-ing system for two model sugar analytes,�-lactose andl-arabinose, was developed based on this principle.Detection limits obtained for both sugars were in themicromolar range. These detection limits are at least acouple of orders of magnitude lower than the detection


Fig. 7. Results of the selectivity study represented in bar graph: (�) induction of BFP2; ( ) induction of GFPuv. The mixture of cellswas induced to final concentrations of 1× 10−3 M of various sugars for 3 h. Fluorescence emission was measured at 443 and 506 nm forBFP2 and GFPuv, respectively. Data are mean± S.D. (n = 3).

limits reported with currently available methods fordetecting lactose [39–41] and arabinose [42]. The re-sponse time of our system is slightly longer than thatof some of the available methods for detecting thesesugars, but it can be reduced by measuring the signal in30 min, which still provides a signal increase of nearly40%. The system developed is highly selective towards�-lactose andl-arabinose. It was demonstrated that avariety of structurally related sugars did not induce anyresponse. Thus, the system developed in this study pro-vides good selectivity and therefore, is a good alterna-tive to existing methods used in the detection of sugars.In addition, this system also contributes in the studyof bioavailability of the analytes. Since the regulatoryproteins sense their corresponding analytes only whenthey are present inside the cell, this system providesthe tool to determine bioavailability of the respectiveanalytes. The cell-based sensing system coupled with areporter protein like GFP provides a convenient meansof detecting analytes without any need for isolatingand maintaining biological components. Furthermore,as GFP and its variants do not require any substrates,their use in cell-based assays offer the advantage ofreal time monitoring of the effects of the analyte onphysiology of the cells. It can be further envisioned

that different mutants of GFP or other reporter pro-teins with differing spectral characteristics can havepotential applications in array detection. The con-tinued discovery of novel reporter proteins throughgenetic engineering and through nature explorationwill undoubtedly have a high impact in multianalytesensing.

Acknowledgements

This work was supported in part by the NationalScience Foundation (Grant CHE-9502299M) and theNational Institutes of Environmental Health (NIEHSwith funding provided by the Environmental Protec-tion Agency (1P42ES07380). SD is a Cottrell Scholarand a Lilly Faculty Awardee. SS thanks the Ken-tucky Research Challenge Trust Fund for a PredoctoralFellowship.

References

[1] J. Frengen, T. Lindmo, E. Paus, R. Schmid, K. Nustad, J.Immunol. Methods 178 (1995) 141.


[2] J.H. Hayes, H.B. Halsall, W.R. Heineman, Anal. Chem. 66(1994) 1860.

[3] S.E. Cowan, E. Gilbert, A. Khlebnikov, J.D. Keasling, Appl.Environ. Microbiol. 66 (2000) 413.

[4] I. Hemmila, S. Holtiinen, K. Pettersson, T. Lovgren, Clin.Chem. 33 (1987) 2281.

[5] J. Vuori, S. Rasi, T. Takala, K. Vaananen, Clin. Chem. 37(1991) 2087.

[6] F.H. Wians Jr., F.H.J. Dev, M.M. Powell, J.I. Heald, Clin.Chem. 32 (1986) 887.

[7] S.M. Gow, G. Caldwell, A.D. Toft, G.J. Beckett, Clin. Chem.32 (1986) 2191.

[8] M. Verhaegen, T.K. Christopoulos, Anal. Chem. 70 (1998)4120.

[9] M.N. Nanjee, N.E. Miller, Clin. Chem. 42 (1996) 915.[10] Y.-Y. Xu, K. Petterson, K. Blomberg, I. Hemmila, H. Mikola,

T. Lovgren, Clin. Chem. 38 (1992) 2038.[11] S.G. Hadfield, A. Lane, M.B. MacIllmurray, J. Immunol.

Methods 97 (1987) 153.[12] U. Narang, P.R. Gauger, A.W. Kusterbeck, F.S. Ligler, Anal.

Biochem. 255 (1998) 13.[13] P. Sticher, M.C. Jaspers, K. Stemmler, H. Harms, A.J.B.

Zehnder, J.R. van der Meer, Appl. Environ. Microbiol. 63(1997) 4053.

[14] Y. Ikariyama, S. Nishiguchi, T. Koyama, E. Lobatake, M.Aizawa, M. Tsuda, T. Nakazawa, Anal. Chem. 69 (1997)2600.

[15] V. de Lorenzo, S. Fernandez, M. Herrero, U. Jakubzik, K.N.Timmis, Gene 130 (1993) 41.

[16] B.M. Applegate, S.R. Kehrmeyer, G.S. Sayler, Appl. Environ.Microbiol. 64 (1998) 2730.

[17] S. Ramanathan, W. Shi, B.P. Rosen, S. Daunert, Anal. Chim.Acta 369 (1998) 189.

[18] S. Ramanathan, W. Shi, B.P. Rosen, S. Daunert, Anal. Chem.69 (1997) 3380.

[19] D.L. Scott, S. Ramanathan, W. Shi, B.P. Rosen, S. Daunert,Anal. Chem. 69 (1997) 16.

[20] S. Tauriainen, M. Karp, W. Chang, M. Virta, Biosens.Bioelectron. 13 (1998) 931.

[21] M. Virta, J. Lampinen, M. Karp, Anal. Chem. 67 (1995) 667.[22] P. Corbisier, D. van der Lelie, B. Borremans, A. Provoost,

V. de Lorenzo, N.L. Brown, J.R. Lloyd, J.L. Hobman, E.Csoregi, G. Johansson, B. Mattiasson, Anal. Chim. Acta 337(1999) 235.

[23] R.S. Shetty, S. Ramanathan, I.H. Badr, J.L. Wolford, S.Daunert, Anal. Chem. 71 (1999) 763.

[24] L. Bousse, Sens. Actuators B 34 (1996) 270.[25] S. Ramanathan, M. Ensor, S. Daunert, TIBTECH 15 (1997)

500.[26] L.U. Colon, R. Dadoo, R. Zare, Anal. Chem. 65 (1993) 476.[27] S. Honda, S. Iwase, A. Makino, S. Fujiwara, Anal. Biochem.

176 (1989) 72.[28] T.W. Garner, E.S. Yeung, J. Chromatogr. 515 (1990) 639.[29] J. Haselhoff, Methods in Cell Biology, Vol. 58, Academic

Press, New York, 1999 (Chapter 9).[30] L.H. Naylor, Biochem. Pharmacol. 58 (1999) 749.[31] A. Crameri, E.A. Whitehorn, E. Tate, W.P.C. Stemmer, Nature

Biotechnol. 14 (1996) 315.[32] R. Heim, R.Y. Tsien, Curr. Biol. 6 (1996) 178.[33] B.P. Cormack, R.H. Valdivia, S. Falkow, Gene 173 (1996) 33.[34] J. Sambrook, E.F. Fritsch, T. Maniatis (Eds.), Molecular

Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY, 1989.

[35] G.C. Gil, R.J. Mitchell, S.T. Chang, M.B. Gu, Biosens.Bioelectron. 15 (2000) 23.

[36] J.C. Lewis, S. Daunert, Anal. Chem. 71 (1999) 4321.[37] J.C. Lewis, A. Feltus, C.M. Ensor, S. Ramanathan, S. Daunert,

Anal. Chem. 70 (1998) 579A.[38] K.S. Matthews, J.C. Nichols, Progress in Nucleic Acid

Research and Molecular Biology, Vol. 58, Academic Press,New York, 1998, p. 127.

[39] M. Filipiak, K. Fludra, E. Gosciminska, Biosens. Bioelectron.11 (1996) 355.

[40] F. Sevilla, T. Kullick, T. Scheper, Biosens. Bioelectron. 9(1994) 275.

[41] J. Svitel, O. Curilla, J. Tkac, Biotechnol. Appl. Biochem. 27(1998) 153.

[42] Q. Hu, T. Zhou, L. Zhang, Y. Fang, Analyst 126 (2000) 298.

Simultaneous detection of analytes based on genetically engineered whole cell sensing systems

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