Fast determination of viable bacterial cells in milk samples using impedimetric sensor and a novel calibration method

Highlights

• Impedance measured at 10 kHz was used to determine microbial growth.

• Changes in impedance are related to Warburg diffusion impedance.

• Impedance changes depend on the number of microorganisms in the sample.

• Method proposed allows to measure E.coli concentration with 5–12% error margin

Abstract

As a first stage toward the development of an impedimetric biosensor to quantify viable bacterial cells, this work defines the relationships between microbial growth and impedance changes associated with acidification of the culture medium. Escherichia coli bacterium was used as a model of a common pathogenic bacterium found in a foodstuff. Impedance was measured at a fix frequency of 10 kHz using interdigitated microelectrodes. Impedance changes were found to depend on the number of microorganisms in a sample. However, as the kinetics greatly depends on the bacterial concentration, it was not possible to obtain a single calibration curve in a wide concentration range at a fixed incubation time. To resolve this, a novel calibration method was proposed by measuring the sensor response at 270 and 390 min of incubation and taking a mean value. Obtained calibration plots were used to determine E. coli in a spiked milk samples. Performed experiments demonstrated the capacity of the method to detect E.coli concentrations in a range between 10² and 10⁶ cfu mL⁻¹ in milk in only 6 hours with 5–12% error margin.

Introduction

Effective pathogen detection is essential for the prevention and treatment of infectious diseases. Additionally, an accurate differentiation of live bacteria is important in many applications. Biosensors based on different types of transducers may be used for highly sensitive and specific detection of pathogens [1]. However, although those permit to reach good detection limits in most cases they are not able to differentiate between viable and non-viable cells. Conventional and standard methods of viability and quantification in bacteria are traditionally associated with the ability to form colonies on agar growth medium and to proliferate in liquid nutrient broths. However, these traditional culture-based tests are time-consuming and are not providing real-time results or timely information that is needed in applications such as industrial manufacturing.

By metabolic pathways bacteria convert large molecules, such as polysaccharides, lipids, nucleic acids and proteins, into charged, smaller and more mobile metabolites. These may include lactic acid, acetic acid, carbon dioxide, ammonia, bicarbonate and urea, the presence of which provokes a change in the ionic composition of the growth media [2]. Thus, these changes can be measured and related to bacterial concentration for determination of microbial growth [3]. Since bacterial metabolism may significantly alter electrical conductivity of the growth media, impedance technique can be successfully applied for estimation of microbial biomass [4], detection of microbial metabolism, as well as for determination of the physiological state of bacteria growth [5], [6], [7]. Advantages of this approach are high sensitivity, relative simplicity and comparatively low cost of the required experimental equipment [8].

Impedance measurements of microbial growth are commonly related to conductivity changes due to the release of ionic products, however it should be noted that most of metabolic products of bacteria are of acidic nature, so that the microbial growth media can also lead to pH changes of the media [8]. Thus, pH measurements can also be used to control the bacteria growth [9].

Monitoring of acidic products concentration is usually carried out by commercial pH electrodes. Conventional glass pH electrodes have been exploited to detect pH changes in the medium produced by bacteria, for example in fermentation processes [10]. However, glass pH electrodes present several drawbacks, such as high price, large size, fragility, and miniaturization and integration constraints. For these reasons, numerous pH sensor alternatives have been explored over the last decades [11], [12]. Among them stand out ion-sensitive field effect transistors (ISFETs) [13] and potentiometric ion selective electrodes based on different metal oxides [14]. One of the drawbacks of potentiometric measurements is that the method requires the use of a reference electrode, which makes the whole system too bulky. It is possible to design small size reference electrodes to be integrated within the system, but these have a short working lifetime and require maintenance. Integrated reference electrode—ISFET pair may be used in a small volume samples but the costs are rather high to be implemented in real life applications.

Potentiometric methods in comparison to conductivity measurements are less sensitive and rapid [15]. However, if the initial conductivity of the broth is high, then conductivity changes will be small and pH in this particular case should be a better indicator of bacterial activity.

From presented it follows that the impedance technique to register changes in a growth medium resulting from the metabolic products released by bacterial may be regarded as a most convenient one. Impedance measurements are carried out by applying a small AC signal between a pair of metal electrodes registering the resulting current and a phase shift between the voltage and the current. Electrochemical impedance spectroscopy (EIS) determines the response of an electrochemical cell to a voltage at different frequencies. Thus, impedance spectrum is obtained allowing characterization of the system composed by electrodes and bulk medium. EIS spectra are often analyzed using an equivalent circuit which consists of resistances and capacitances combined in parallel or serially, as required. The most typical equivalent circuit for this type of measurements can be represented by a series combination of a bulk solution resistance and an interfacial capacitance at the electrode-solution interface.

Electrodes for impedance measurements may be arranged as a pair of oppositely placed electrodes. However, it is possible to miniaturize this electrode arrangement by using two in-plain interdigitated microelectrodes. These impedimetric transducers, called interdigitated electrode array (IDEA), has been demonstrated as a promising for monitoring the growth of bacteria [7], [16], [17], since they present advantages in terms of fast establishment of the steady state signal, increased signal-to-noise ratio and the use of small sample solution volumes.

In this work to miniaturize the system instead of a pH-sensor and reference electrode an impedimetric sensor based on an interdigitated electrode array [18] was used to detect metabolic activity of Escherichia coli bacteria. Escherichia coli was chosen as a target because it is an important pathogen responsible for numerous water and food transmitted infections whose growth under aerobic conditions causes the formation of acidic by-products, of which acetate is the most predominant [19], [20].