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This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies areencouraged to visit:
a Department of Physical Electronics, School of Electrical Engineering, Faculty of Engineering, Tel Aviv University, Ramat Aviv, Tel-Aviv 69978, Israelb Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israelc Division of Qualitative Hydrology, Federal Institute of Hydrology (BfG), Koblenz 56068, Germany
a r t i c l e i n f o
Article history:Received 31 October 2008Received in revised form 31 December 2008Accepted 22 January 2009Available online 31 January 2009
This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The biosen-sor presented here is based on bacterial cells that are genetically “tailored” to generate a sequence ofbiochemical reactions that eventually generate an electrical signal in the presence of genotoxicants. Thebacterial assay was affected by toxicant contaminated water for an induction time that ranged between30 min and 120 min. Enzymatic substrate (pAPP) was added to the assay generating the electrochemi-cal active material (pAP) only when toxicants are sensed by the bacteria. The bacteria were integratedonto a micro-chip that was manufactured by MEMS technology and comprises various micro-chamberswith volume ranging between 2.5 nl and 157 nl with electrode radius between 37.5 �m and 300 �m. Wedescribe the biochip operation, its electrochemical response to calibration solutions as well as to thewhole cell assays. The potential use of the whole cell biochip for toxicity detection of two different geno-toxicants, nalidixic acid (NA) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), is demonstrated. Wedemonstrate minimal toxicant detection of 10 �g/ml for NA using 30 min for induction and 0.31 �M forIQ using 120 min for induction, both 3 min after the addition of the substrate material.
Integration of various chemical devices and complex operationsonto a micro-chip, which is often referred to as a micro-total anal-ysis system (�-TAS) or “lab on a chip”, is currently generatingmajor interest due to the promising characteristics of such sys-tems. Over the last few years, the dimensions of micro-chip areahave decreased, resulting in the ability to accommodate sensorybiological cells on solid-state platforms with micron scale features.This, in turn, has enabled to harness biological cell-based reactionsby incorporating various types of microbial cells into micro-environmental systems [1]. In such biotic-microelectromechanicalsystems (biotic-MEMS) sensors, the main sensing element is themicrobial cell that converts a chemical or a biological signal toan electrical output [2]. Microbial sensors are less sensitive thanenzyme-based sensors to environmental changes, however, can begenetically modified, by using relatively simple genetically engi-neering methods, to respond [3] and detect a very complex seriesof reactions that can exist only in an intact, functioning cell [4].Microbial biosensors have been utilized for environmental moni-
toring [5,6], food monitoring [7–9], monitoring of microbial growthrate [10] and biocide measurements [11].
There are few methods to detect the generated signal from themicrobial cells, e.g. optical, electrochemical, electrical and mechan-ical. In this work we describe an electrochemically based platform.Electrochemical biosensors are based on a bio-interaction pro-cess where electrochemical species are consumed or generatedproducing a measurable electrochemical signal. Electrochemicalmeasurements detect only the electrical properties of the analytespecies undergoing redox reactions, therefore, they are limited tosensing electro-active species. Electrochemical detection usuallyuses amperometry, potentiometry or conductometry [12]. Amper-ometric techniques are linearly dependant on the electro-activespecies concentration. Amperometry is applied in our case sincewe generate current and it gives useful dynamic range and signal tonoise ratio [13–19]. Potentiometric biosensors [20–25] measure thepotentials at the working electrode in respect to the reference elec-trode. They can be applied in our case although they generate a verysmall signal. Conductometric biosensors measure the impedance atthe chip level at various frequencies. Therefore, it detects changesin the biological system in response to the toxic material [17,26–31].
Our goal, as presented here, is to develop and integrate a novelwhole cell micro-fluidic biochip for electrochemical water tox-icity detection harboring whole cell electrochemical biosensors.The manufactured micro-chip was characterized and calibrated
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Fig. 1. (A) Silicon-based micro-chip comprises four differentially sized electrochemical micro-chambers. (B) Inside view of a single three-electrode electrochemical micro-chamber. (C) Schematic layout of the electrochemical unit.
by conventional electrochemical assays, demonstrating its func-tionality. The potential toxicity detection of the biochip wasexamined with two genotoxicants; nalidixic acid (NA) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). NA is presented here as amodel genotoxicant. IQ is a genotoxic substance that is a highly rel-evant environmental pollutant [32]. Genotoxicants in general are ofimportance because of their specific DNA-damage inducing effect,which is potentially irreversible. These toxicants are used to drivethe genetically engineered bacteria to generate a measurable elec-trochemical bio-signal. The output current was measured and wepresent it as is or following simple signal processing showing goodsignal to noise ratio.
2. Experimental
2.1. Platform fabrications
A portable solid-state system for whole cell electrochemicalanalysis was made using conventional micro-fabrication and inte-gration processes. The basic unit of the system is an electrochemicalunit made of two micro-chips (Fig. 1C): (a) the electrodes chip (partA) and (b) the micro-fluidics part (part B). The electrochemical chip(Fig. 1A and B) includes micro-chambers fabricated on single crystalsilicon substrate by a deep-etch MEMS process which is describedin Ref. [33]. The electrochemical chip comprises of four cylindricalelectrochemical 50 �m deep micro-chambers with different radii:1 mm, 0.5 mm, 0.25 mm, and 0.125 mm. The corresponding vol-umes were 157 nl, 39 nl, 9.8 nl, and 2.5 nl. Each chamber containsthree electrodes: working electrode (WE), counter electrode (CE)and reference electrode (RE). The electrodes are made of thin evap-orated gold (200 nm)/Cr(15 nm). The open reference electrode wascoated with Ag/AgCl layers (Fig. 1B). The Ag/AgCl open referenceelectrode was manufactured by a two-step electrochemical pro-cess [33]: (a) Ag electroplating (standard Ag nitrate bath) at a rate of0.57 �m/min. (b) Anodization of the Ag in a bath containing chlorineions.
The second part of the system, the micro-fluidic chip, wasmade from polydimethylsiloxane (PDMS) and it includes the micro-channels feeding the water to the electrochemical Si-based part.The chip was manufactured using conventional molding of PDMSinto a machined Brass matrix. The PDMS micro-fluidic chip wasmounted on the Si-based electrochemical chip and both were pack-aged on a measurement platform made of Delrin and a Perspexseal. The Brass matrix, the Perspex seal and the Delrin platformwere produced by Computer Numerical Control (CNC) machin-ing (Fig. 2) with a minimum resolution of about 50 �m. Theelectrochemical micro-chambers and the chip mounted on themeasurement platform were connected to a portable potentiostat(EmStat, made by PalmSens Inc.) using a 16 channel multiplexerallowing a sequential reading and monitoring of four electrochem-ical micro-chips (each with four micro-chambers) at the sameexperiment.
The micro-chip was evaluated by testing its electrochemical per-formance for the analysis of a known chemical reaction. Therefore,ferrocyanide (0.010 M), ferricyanide (0.010 M) and KCl (1 M) weremixed yielding a solution with Fe2+/Fe3+ ions. This assay was eval-uated by conventional cyclic voltammetry.
The second test was for the evaluation of the oxidationof the reaction by products. We intentionally introduce para-aminophenol (pAP, FW 145.6, Sigma) into deionized water (DI) andmeasure the electrochemical activity of the pAP under conditionssimilar to that of those on the real test chip. The pAP was dilutedto final concentrations of 0.4 mg/ml, 0.04 mg/ml and 0.004 mg/ml.The tested aliquots volume was 2.5 �l that were introduced into theelectrochemical micro-chamber. Chrono-amperometry at appliedpotential of 300 mV was measured using the EmStat (made byPalmSens) potentiostat. The data was stored for further process-ing using a universal serial bus interface (USB) between the EmStat(made by PalmSens) and a conventional PC.
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Fig. 2. (A) Layout of the measurement platform comprises Perspex seal, micro-fluidic PDMS molding, and electrochemical micro-chip. (B) The real micro-fluidic electrochem-ical chip system.
2.3. Toxicity bio-detection assays
Toxicity analysis experiments utilized two strains of geneti-cally engineered bacteria; Escherichia coli (E. coli) for the nalidixicacid (NA) detection, and Salmonella typhimurium TA1535 (S.typhimurium) for the 2-amino-3-methylimidazo[4,5-f]quinoline(IQ) detection. E. coli RFM443/pBR2TTS cells harboring a sulA::phoAfusion were grown overnight in MOPS [34] growth medium con-taining 0.1 mg/ml ampicillin with shaking conditions at 37 ◦C. Theovernight culture was diluted 1/100×, regrown to an optical densityof 0.2 (600 nm). Finally NA (FW 254.22, Sigma) was added to a finalconcentration of 10 �g/ml. Following further incubation at varioustimes of 0 min, 30 min, 60 min, 90 min, or 120 min under similarconditions, aliquots of 3 �l were introduced into the electrochem-ical micro-chambers. Shortly after applying a constant potentialof 300 mV vs. Ag/AgCl in the electrochemical chamber, the enzy-matic substrate para-aminophenyl phosphate (pAPP, MW 211.09,diagnoSwiss) was added reaching a concentration of 0.8 mg/ml.
Control samples were prepared with the addition of growthmedium instead of NA or pAPP to the incubation stage in orderto verify the influence of NA on the induction of the bacteria andpAPP on the generated bio-electrochemical response.
S. typhimurium TA1535 pSK1002 [35] cells were used for theIQ bio-detection. The exposition of bacteria was done accordingto Reifferscheid et al. [36]. An overnight culture of the bacteriawas refreshed in 20 ml TGA-medium (1:10 dilution of bacteria)and grown for further 2 h (37 ◦C, 150 rpm). Prior to exposition theoptical density (� = 595 nm) of the bacterial culture was adjustedto 0.44 by dilution with TGA medium. The pre-genotoxic com-pound IQ was activated by adding a post-mitochondrial liverhomogenate of induced rats (S9-fraction). The S9-fraction wasobtained from RCC Cytotest Cell Research GmbH (Roßdorf, Ger-many). It was stored at −80 ◦C and kept on ice after thawing forimmediate usage. Cofactors and salts were added for the activa-tion of S9-enzymes. The final concentrations of the componentsin the reaction mixture were S9-fraction 0.8% (v/v), NADP 2.9 mM,glucose-6-phosphate 3.3 mM, KCl 24 mM and MgCl2 5.9 mM. 70 �lof the bacterial suspension containing the S9-fraction (240 �l S9-fraction in 8 ml bacterial culture OD595 = 0.44) were added to 20 �lof a stock solution (containing the cofactors and salts in 10× TGAmedium) and mixed with 500 �g/ml ampicillin. Finally, 180 �lof sample (aqueous dilutions of an IQ-stock solution in DMSO,final IQ-concentrations 5 �M, 1.25 �M and 0.31 �M) or negativecontrol (3% DMSO in water) were added yielding a total vol-ume of 270 �l. The OD595 of the exposed mixture was measuredafter an incubation time of 2 h at 37 ◦C and 900 rpm in shakingconditions.
The activity of the reporter enzyme �-galactosidase in theinduced bacteria was measured by making use of the sub-strate para-aminophenyl �-d-galactopyranoside (pAPG, 0.5 mMfinal concentration, Sigma). Shortly after the addition of pAPG,aliquots of 3 �l were introduced into the electrochemical micro-chambers and a constant potential of 300 mV vs. Ag/AgCl wasapplied on the electrochemical chamber in order to quantify theenzymatically generated product pAP. The setup was connected insuch a way that the output current was monitored (i.e. amperom-etry measurement).
3. Results
In this section we present the results of the measurements of themicro-fluidic whole cell electrochemical biochip. We first presentthe calibration and the testing schemes and later the biochipresponse to two toxicants: NA and IQ as aforementioned in theprevious section.
3.1. Electrochemical characterization of the micro-chip
The electrochemical activity of the manufactured micro-chipwas validated by a cyclic voltammetry assay with a Fe2+/Fe3+
electro-active solution. The cyclic voltammograms that resultedfrom the differently sized electrochemical micro-chambers areshown in Fig. 3A. The voltammograms using the electrochemi-cal micro-chip show clearly the cathodic reduction of the Fe3+
and the anodic oxidation of the Fe2+. The measured open cir-cuit potential of the reaction (e.g. 0.188 V vs. Ag/AgCl referenceelectrode) approximately corresponded to the predictable halfcell potential (e.g. 0.124 V vs. Ag/AgCl reference electrode) of theferrocyanide–ferricyanide reaction according to Nernst equation.The deviation of the experimented open circuit potential may bedue to the fact that an open Ag/AgCl reference electrode was usedwhen its half-cell potential was slightly influenced by the chem-ical reaction in the cell. The peak current of the anodic currentand the associated applied potential (vs. the open Ag/AgCl elec-trode) were extracted and plotted vs. the area and the 1/area of theworking electrode, respectively (Fig. 3B and C). The anodic currentpeak plot (Fig. 3B) yields a positive linear relation with a slope of23.2 ± 1.7 �A/mm2. This relation meets the expected relation (e.g.15.1 �A/mm2 at 25 ◦C) calculated from Bard and Faulkner [37]. Fur-thermore, the applied potential at the anodic peak as a function ofthe 1/area of the working electrode demonstrated a positive lin-ear relation (Fig. 3C). This dependence may be attributed to thefact that Nernst equation is slightly modified on very small elec-trodes, when a current flow may cause an increase with the ohmic
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Fig. 3. (A) Cyclic voltammograms resulted by a Fe2+/Fe3+ assay with the differently sized electrochemical micro-chambers, v = 50 mV/s. (B) Influence of the area of theworking electrode on the anodic current peak. (C) Influence of the 1/area of the working electrode on the applied potential at the anodic peak.
drop (uncompensated resistance and solution resistance) near theelectrode.
The electro-active species pAP is the product molecule producedby the biochemical reaction of the enzyme alkaline phosphataseusing pAPP as a substrate [38] or the enzyme �-galactosidase usingpAPG as a substrate [39]. The induction level of the enzyme bythe genetically engineered bacteria is proportional to the toxi-cant concentration in the sample. Cyclic voltammetry assays ofdifferent scan rates were applied on duplicate electrochemicalmicro-chambers with aliquots of 0.4 mg/ml pAP solution. Theanodic current peak was measured and is plotted as a functionof the square root of the scan rate (Fig. 4). The result is a lineardependence which fits conventional cyclic voltammetry modeling[37]. Furthermore, a correlation coefficient of 0.98 was calculatedbetween the two similar electrochemical micro-chambers indi-cated linear dependence. Therefore, these results demonstrate thereproducibility of the electrochemical signal in the micro-chip.
Fig. 4. Impact of the square root of the scan rate at a cyclic voltammetry assay onthe peak of the anodic current in the presence of 0.4 mg/ml pAP, measured in twosimilar electrochemical micro-chambers.
The reactivity of the electro-active species pAP was tested bythe micro-chip. Chrono-amperometry results for the response todifferent concentrations of pAP are shown in Fig. 5. The resultsdemonstrated the dependence of the generated electrochemicalcurrent as a function of the electro-active species concentrations. Itis important to point at the signal dependence on the electro-activespecies dose. This dependence is important since this species, i.e.pAP, is the end product of the biochemical reactions in the pre-sented bacterial biosensor, hence its production rate is expected todepend on the toxicant concentration in the analyzed sample.
3.2. Electrochemical bio-detection of toxic materials
The presence of toxicants induces a cascade of biological reac-tions in the genetically engineered bacteria producing an increased
Fig. 5. Chrono-amperometric detection of different concentrations of pAP by themicro-chip.
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Fig. 6. (A) Chrono-amperometric results of bacterial cells following 1 h of incubation in the presence and the absence of NA. (B) Chrono-amperometric results of bacterialcells following increasing periods of induction time with NA. (C) Impact of the induction time with NA on the slope of the detected electrochemical current.
concentration of the enzymatic bio-reporter alkaline phosphatase.This enzyme catalyzes the reaction converting the substrate pAPPto the electro-active species pAP. Therefore, by using an appropriateelectrochemical transducing system, the generated electrochemicalbio-signal can be detected. Fig. 6A presents chrono-amperometricresults of the response of E. coli bacteria in the presence and theabsence of the model toxicant NA. The response of the bacteriain the presence of NA showed an increasing electrochemical cur-rent after pAPP was added. This is compared to the response ofinduced bacteria without the addition of pAPP which demonstrateda decreasing current characteristics. Furthermore, the inducedbacteria exhibited a more rapidly increasing electrochemical cur-rent than non-induced bacteria when pAPP was added to bothsamples.
Note that the “non-induced” (i.e. not exposed to the toxicant)bacteria demonstrated a unique current rise when pAPP was addedto the bacterial suspension. That result was reproducible and ischaracteristics to that specific bacterial system and toxicant. It is
suggested that the reason for these characteristics may be due tothe toxic effect on the bio-physiological state of the bacterial cellsfollowing an incubation period with the toxicant.
Chrono-amperometric results of bacterial cells following differ-ent induction periods with NA are shown in Fig. 6B. The resultsdemonstrated the dependence of the generated electrochemicalcurrent with the total induction time of the bacteria by the toxi-cant. For longer induction times the current increased more rapidly(Fig. 6C), indicating the ongoing enzyme production in the bacterialcells during the induction and hence an increasing enzyme con-centration with time. Therefore, increasing induction time yieldshigher alkaline phosphatase concentration yielding higher gen-eration rate of pAP. Since the current is proportional to the pAPconcentration, an increasing pAP generation rate yields an increas-ing electrochemical current. This result was verified and modeledfor a different system [40] where the enzyme was �-galactosidaseand the substrate was also pAPG similar to the work that is pre-sented here.
Fig. 7. (A) Impact of the IQ concentration on the differential slope of the electrochemical current. (B) Impact of the radius of the working electrode on the slope of theelectrochemical current.
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Fig. 8. Induction factor values for two different size electrochemical micro-chambers in terms of the working electrode radius.
The impact of the genotoxic material IQ on the electrochemi-cal bio-signal was tested and analyzed with the micro-chip. Thedependence of the differential slope (the difference between theslope detected by induced bacteria and the slope detected by non-induced bacteria) on the IQ concentration is shown in Fig. 7A.Higher IQ concentrations demonstrated more rapidly increasingelectrochemical current characteristics. The influence of the WEradius on the slope of the detected electrochemical current is shownin Fig. 7B. As expected, the current increased with the dimensionof the micro-chambers. However, larger micro-chambers exhibitedalso a larger current from non-induced bacterial cells (negativecontrol). Therefore a figure of merit describing the bio-detectionefficiency was devised in order to quantify the system signal(response to induced bacterial cells) to noise (response of non-induced bacterial cells) performance.
The bio-detection efficiency of the micro-chip was modeled bydefining induction factor (IF) values. The induction factor is the ratiobetween the bio-signal detected from bacterial cells in the presenceof a toxicant and the bio-signal detected from bacterial cells in theabsence of a toxicant (negative control). Induction factor values fortwo different size electrochemical micro-chambers are shown inFig. 8. Both cells show similar (within experimental error) induc-tion factor. These results support the claim that scaling down of themicro-chip is possible and it will not, until certain limit, deterioratethe induction factor.
4. Conclusions
A novel micro-fluidic whole cell biosensor, integrating fourbiochips with four micro-chambers each, is demonstrated. Thebiosensor was characterized by standard electrochemical assaysand its electrochemical characteristics are described. The ability ofthe biosensor to analyze toxicity is demonstrated with the detec-tion of both model toxicant NA and genotoxic material (IQ). Thebiochip performance elucidated the dependence of the bio-signalas a function of the induction time of the bacterial biosensor withthe toxic material.
The ability to perform multiple assays on one chip combinedwith the promising characteristics of whole cell biosensors allowsfunctional screening of numerous unknown analytes. Scaling downthe architecture of the micro-chip is one way to achieve thisgoal. Therefore, the impact of the dimensions of the electrochem-
ical chamber on the electrochemical bio-signal is described. Byminiaturizing the size of the chamber, the diffusion distance ofelectro-active species to the electrode also decreases, allowing ana-lyzing nano-liters of samples in a rapid and sensitive detection byelectrochemical biosensors.
Acknowledgements
This work was supported by the “Dip-chip” project funded bythe German–Israeli BMBF-MOST Cooperation in Water Technologygrant number WT0601/02WU0844. The work was also supportedby the Tel Aviv University Scholarship Fund. We wish to thank Mr.Nick Fishelson, Mr. Alexander Gurevitch, Ms. Danna Landau, Ms.Ma’ayan Gal-On, Mr. Doron Albert and Mr. Ezra Shaked for the use-ful discussions and lab assistance. The devices were fabricated atthe Tel Aviv University Micro and Nano Characterization and Fabri-cation facility (MNCF).
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