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Bacterial concentration detection inwater by microfabricated impedance

biosensor

Marco Grossi marco.grossi8@unibo.it, Bruno Riccò - Department of Electronic Engineering(D.E.I.S.), University of Bologna, Bologna

Daniele Gazzola, Manuele Onofri, Giampaolo Zuccheri - Health Sciences and Technologies(HST-ICIR), University of Bologna, Bologna

Diego Matteuzzi - Department of Pharmaceutical Sciences, University of Bologna

SummaryControl of water microbial content is of great importance to guarantee the absence of pathogens.The bacterial concentration is traditionally measured by standard plate count, a technique that isreliable but characterized by long response time and must be performed in microbiology laborato-ry with the aid of trained personnel. The impedance technique, that measures the bacterial con-centration by analyzing the sample electrical characteristics, is competitive with the standard tech-nique since features shorter detection times (3 – 12 hours vs. 24 – 72 hours of plate count) and canbe easily realized in automatic form. The present work shows a microfabricated sensor featuringgold electrodes (1mm2 area separated by 100ìm) used to measure the concentration of a wild typecoliform strain (isolated in river water). The presented sensor is capable to detect high microbialconcentration (106 cfu/ml) in relatively short time (225 minutes) and, compared to other imped-ance biosensors, has the advantage to properly work at higher frequencies (extending the workingfrequency range to over 1 MHz) with benefits for measure reliability.

RiassuntoIl monitoraggio della contenuto microbico delle acque è di grande importanza al fine di garantirel’assenza di microorganismi patogeni. La concentrazione batterica viene determinata tramite con-ta in piastra, tecnica che risulta affidabile ma richiede tempi lunghi e può solo essere effettuata inlaboratori di microbiologia da personale qualificato. La tecnica impedenziometrica, che valuta laconcentrazione microbica tramite l’analisi delle caratteristiche elettriche del campione, è una tec-nica competitiva con quella tradizionale in quanto garantisce tempi di risposta più brevi (3 – 12ore rispetto a 24 – 72 ore della conta in piastra) ed è facilmente automatizzabile. Nel presentelavoro viene mostrato un sensore micro fabbricato con elettrodi in oro (di dimensione 1mm2

separati da una distanza di 100ìm) utilizzato per la determinazione della concentrazione di uncoliforme isolato nelle acque di fiume. Il sensore si dimostra capace di rilevare alte concentrazionibatteriche (106 cfu/ml) in tempi relativamente brevi (225 minuti) e ha il vantaggio rispetto adaltri biosensori impedenziometrici di poter operare a frequenze più elevate (fino a 1 MHz) connotevoli benefici per quanto riguarda l’affidabilità della misura.

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1. IntroductionBacterial concentration detection is of great importance in environmental monitoring and ex-istent national and international regulations guarantee water quality and safety [1]. Environ-mental waters (such as river waters and seawaters) are periodically screened to ensure thatmicrobial concentration is within legal limits and that pathogens that can endanger humanhealth are absent. Usually this is obtained by screening the samples for microorganisms thatare related to faecal contamination [2]: in fact, from a statistical standpoint, these present agood correlation with the presence of pathogens. Traditionally, coliforms are considered thebest indicators of faecal contamination [3] since they originate in the intestine of warm blood-ed animals.Water bacterial concentration is usually measured by Standard Plate Count (SPC) technique[4], a reliable method but characterized by long response time (24 – 72 hours depending onthe screened microbial strain) and by the need to be performed in microbiology laboratorieswith the aid of trained personnel. This prevents SPC to be used for fast in-situ detection ofbacterial contamination.In the last years many innovative techniques have been proposed for microbial concentrationdetection based on transduction methods such as amperometry [5], bioluminescence [6], tur-bidity [7], piezoelectricity [8] and impedance [9]. A set of instruments for coliforms detectionin water are produced by IDEXX (Westbrooke, Maine, USA). Colilert, Colilert 18 and Col-isure [10][11] are based on the coliform property to produce â-glucuronidase as a result oftheir methabolism. The IDEXX instruments are however laboratory oriented and the timeneeded to measure the microbial concentration is only slightly shorter than SPC.The impedance technique based on classic impedance microbiology [12] is highly competitivewith SPC because it features singnificantly shorter detection times (3 – 12 hours vs. 24 – 72hours) and is easily implementable in automatic form with the possibility to be realized as anembedded portable system for in-situ measurements. The impedance technique works as fol-lows: the sample, eventually diluted in a suitable enriched media, is stored at a temperaturethat favors bacterial growth and its electrical characteristics (the resistive and reactive compo-nents of the impedance Z) are measured at time intervals of 5 minutes. Until the sample bacte-rial concentration is lower than a critical threshold concentration (107 cfu/ml), the electricalparameters are essentially constant (baseline value), while when it exceeds this concentration|Z| begins to decrease (as well as its resistive and reactive components). The time needed toproduce a variation of the monitored electrical parameters is called Detect Time (DT) and isknown to be linearly related to the logarithm of initial bacterial concentration.Different commercial instruments exist that are based on the impedance technique: Bactome-ter by Vitek Systems Ltd (Basingstoke, UK), Malthus by Malthus Instruments Ltd (Bury, UK),Bac Trac by Sy-Lab (Purkensdorf, Austria) and RABIT by Don Whitley Scientific (Shipley,UK). Recently, an embedded portable biosensor system based on the impedance technique[13] has been proposed that is particularly suited for in-situ bacterial screening. All the pre-sented instruments feature stainless steel or platinum as the electrodes material, the inter-elec-trodes distance is in the mm range and a capacity of 3 to 10 ml for the sample under test (SUT)is used.In this work we test a microfabricated sensor, featuring small (1 mm2) gold electrodes separat-ed by 100 ìm, and compare its performance with those of the aforementioned instruments. Theresults indicate that, even if response time is comparable with that obtained in [13], the micro-fabricated sensor features a broader working frequency range, thus allowing for more realiblemeasurements.

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2. ReportThe proposed sensor has been tested using the experimental setup shown in Fig. 1 (a). Thesensor and SUT are stored in a thermal incubator set at 37 °C and the electrical characteristicsare measured by an LCR meter Agilent E4980A. A Laptop PC system controls the LCR metervia USB interface and acquires the measured data for graphing and data filing.The sensor, shown in Fig. 1 (b), features 18 gold microelectrodes and, for the measures, 4electrodes of the top row have been used shorted in couples as shown in Fig. 1 (b). The elec-trodes consist of 1mm2 gold coated area separated by 100ìm. To measure the electrical charac-teristics, the sensor has been stimulated with a sinusoidal test voltage of amplitude 10 mV

PP

and frequency in the range 20 Hz – 2 MHz (logarithmically spaced).

The enriched medium used to favor bacterial growth is Lauria Bertani (modified to feature lowsalt content). The medium composition (for 1 liter of distilled water) is as follows: Tryptone10.0 g, Yeast Extract 5.0 g (pH 7.0). A wild coliform strain (isolated from river water) has beencultured in Lauria Bertani, diluted in different ratios so to obtain different bacterial concentra-tion and inoculated in the medium. The sensor was then immersed in direct contact with theSUT and both stored in the thermal incubator.The equivalent electrical circuit used to model the sensor immersed in the SUT is shown in Fig.2 (a): R

i represents the interface resistance of the electrodes, Cpar the electrodes capacitance due

to the sensor AlN substrate, Rm the medium resistance and Z

CPE the impedance of a constant

phase element (CPE) modeling the non-ideal capacitive electrode-electrolyte interface. As the

Fig. 1 – (a) Scheme of the experimental setup used in the bacterial concentration measurements. The system

features a thermal incubator to store the sample under test, an LCR meter to measure sample electrical

characteristics and a laptop PC for data graphing and filing. (b) Microfabricated sensor used to detect bacterialconcentration. It features 1mm2 gold electrodes separated by 100ìm. 1: Aluminium Nitride (AlN), 2: gold covered

with AlN, 3: gold surface

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bacterial population grows in the SUT, microbial methabolism transforms uncharged com-pounds in highly charged ones, thus increasing the medium ions concentration and the electri-cal conductivity (producing R

m decrease). Moreover, since the ions are subjected to different

forces if near the electrodes or in the bulk of the electrolyte, a double layer is built at theelectrode-electrolyte interface that increases the non-ideal interface capacitance (Q).The parameters of the equivalent circuit that best-fit the measured data have been calculatedusing Multiple Electrochemical Impedance Spectra Parametrization (MEISP) v. 3.0 by Kum-ho Chemical Laboratories.In Fig. 2 (a) and (b) the measured data (dash lines) for |Z| and Arg(Z) as well as the simulatedcurves resulting from the fitted parameters (dot lines) are plotted versus the frequency of thesinusoidal test signal. As can be seen, the simulated curves properly match the measured ones,thus validating the proposed electrical model.

Fig. 2 – (a) Electrical circuit used to fit the measured data. Curves for both measured (dash lines) and simulated

(dot lines) data for |Z| (b) and Arg(Z) (c) are plotted vs. the frequency of the sinusoidal test signal

Measures using the experimental setup of Fig. 1 (a) have been carried out with different con-centrations of the inoculated coliform strain (from 10 cfu/ml to 107 cfu/ml). Although bacteri-al methabolism affects both the bulk conductivity and the interfacial capacitance (i.e. R

m and

Q), the measured values of Q resulted in poor repeatability and low signal-to-noise ratio, re-sulting in low realibility in DT calculation. Thus, in the following, only values of R

m are consid-

ered.In Fig. 3 (a) the percent decrease of R

m, i.e. [(R

m,baseline – R

m)/R

m,baseline]’”100, is plotted vs. time

for two samples characterized by different values of bacterial concentration. As can be seen,the sample featuring lower bacterial concentration (102 cfu/ml) results in higher DT (460 min-utes) than the highly contaminated sample (106 cfu/ml) that features a DT of 225 minutes. Thisshows how the medium resistance R

m can be effectively used to discriminate between different

levels of bacterial concentration.However, since the measure of medium resistance R

m requires a multi-frequency approach (i.e.

the measure of the electrical parameters on a broad range of frequencies and best-fit of themeasured data with the equivalent electrical circuit using a suitable numerical algorithm) we

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have investigated if a single-frequency measure can reliably estimate the sample bacterial con-centration.In Fig. 3 (b) the percent decrease of |Z| is plotted vs. time for the sample characterized by abacterial concentration of 102 cfu/ml and four different frequencies of the sinusoidal test signal(200 Hz, 2 kHz, 20 kHz, 200 kHz). As can be seen, higher frequencies result in more reliabledata, with curves characterized by more stable baseline and thus more accurate determinationof DT. Data on higher contaminated samples (106 cfu/ml) confirmed the same results withmeasured values of DT comparable with those obtained in multi-frequency approach and morereliable results at frequencies higher than 20 kHz.The results show how the proposed microfabricated sensor can broaden the working frequen-cy range. In fact, all the benchtop instruments discussed in the introduction as well as theportable biosensor system in [13] are characterized by a maximum working frequency nothigher than 10 kHz, while the proposed sensor broadens this limit to over 1 MHz, with bene-fits in terms of higher signal-to-noise ratio, more stable baseline and more accurate DT calcu-lation.

Fig. 3 – (a) Percent decrease of Rm plotted vs. time for two samples characterized by different bacterial

contamination. Higher contaminated samples feature lower values of DT. (b) Percent decrease of |Z| plotted

vs. time for single frequency measurements. Higher frequency measurements result in more stable baselineand more accurate DT calculation

3. ConclusionsBacterial concentration detection is very important in environmental monitoring since the pres-ence of pathogens can endanger human health. Bacterial concentration is usually determinedby Standard Plate Count (SPC) technique, a reliable method that is however characterized byslow response (24 – 72 hours depending on the monitored bacteria) and needs a laboratoryenvironment with skilled personnel.The impedance technique for microbial concentration detection is very competitive with SPCsince it features shorter response time (3 – 12 hours depending on the sample bacterial con-tamination) and can be easily automatized and implemented as a portable biosensor system forin-situ measurements.The proposed microfabricated impedance sensor is adequate in bacterial concentration detec-tion since it can measure microbial concentration with response time comparable with thatobtained by benchtop commercial systems (225 minutes for a contamination of 106 cfu/ml).Moreover, the small dimensions (1mm2 electrodes separated by 100 ìm) make it possible to testa small quantity of sample and the working frequency range for the sinusoidal test signal isgreatly improved (maximum frequency 1 MHz) compared to the other systems (maximumfrequency 10 kHz) with benefits in terms of higher signal-to-noise ratio, more stable baselineand more accurate Detect Time calculation.

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edition), New York, 1999.[2] Meays C. L., Broersma K., Nordin R., Mazumder A., “Source tracking fecal bacteria in water: acritical review of current methods”, Journal of Environmental Management, 2004, Vol. 73, 71-79.[3] Romprè A., Servais P., Baudart J., de Roubin M. R., Laurent P., “Detection and enumeration ofcoliforms in drinking water : current methods and emerging approaches”, Journal of MicrobiologicalMethods, 2002, Vol. 49, 31-54.[4] Kaspar C. W., Tartera C., “Methods in Microbiology”, Grigorova & J.R. Norris ed., London: AcademicPress, 1990, Vol. 22, 497-531.[5] Perez F., Tryland I., Mascini M., Fiksdal L., “Rapid detection of escherichia coli in water by a culturebased amperometric method”, Analytica Chimica Acta, 2001, Vol. 427, 149-154.[6] Stanley P.E., “A review of bioluminescent ATP techniques in rapid microbiology”, Journal ofBioluminescence and Chemiluminescence, 2005, Vol. 4 (1), 375-380.[7] Koch A. L., “Turbidity measurements of bacterial cultures in some available commercial instruments”,Analytical Biochemistry, 1970, Vol. 38 (1), 252-259.[8] Kim N., Park I.-S., Kim D.-K., “Characteristics of a label-free piezoelectric immunosensor detectingPseudomonas aeruginosa”, Sensors and Actuators B, 2004, Vol. 100, 432-438.[9] Yang L., Bashir R., “Electrical/electrochemical impedance for rapid detection of foodborne pathogenicbacteria”, Biotechnology Advances, 2008, Vol. 26, 135-150.[10] Cowbum J. K., Goodall T., Fricker E. J., Walter K. S., Fricker C. R., “Preeliminary study on the useof Colilert for water quality monitoring”, Letters in Applied Microbiology, 1994, Vol. 19 (1), 50-52.[11] Chao K. K., Chao C. C., Chao W. L., “Evaluation of Colilert 18 for detection of coliforms and Escherichiacoli in subtropical freshwater”r, Applied and Environmental Microbiology, 2004, Vol. 70 (2), 1242-1244.[12] Firstemberg-Eden R., Eden G., “Impedance microbiology”, New York, Wiley, 1984, Vol. 3, 154-196.[13] Grossi M., Lanzoni M., Pompei A., Lazzarini R., Matteuzzi D., Riccò B., “An embedded portablebiosensor system for bacterial concentration detection”, Biosensors and Bioelectronics, 2010, Vol. 26, 983-990.