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Electrochemical Assays for Microbial Analysis: How Far They Are from Solving

Microbiota and Microbiome Challenges

Elena E. Ferapontova*

Interdisciplinary Nanoscience Center (iNANO), Aarhus University Gustav Wieds Vej 14, DK-8000

Aarhus C, Denmark

E-mail: elena.ferapontova@inano.au.dk

Bacterial sensors are indispensable in environmental monitoring, analysis of food and drink safety,

prevention and treatment of pathogenic infections, antibiotic resistance screening, in combatting

biocorrosion and in biodefense. Recent discoveries within Human Microbiome Project disclosed vital

bacteria’s role in human health and disease prognosis and treatment; they also placed in focus new

analytical tools for bacterial analysis. Here, I discuss several basic concepts underlying the

electrochemical bacterial biosensors: metabolic sensors, biosensors for DNA and RNA extracted from

bacterial cells, and whole bacterial cell sensors, and their contribution to practically sought solutions

for bacterial analysis. Current analytical issues and perspectives are outlined.

Keywords

Bacterial sensors, Pathogen analysis, Electrochemical sensors, Metabolic biosensors,

Electrochemical ELISA, Whole cell analysis

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I. Introduction

Fast, sensitive and inexpensive sensors for bacterial detection are indispensable for environmental

monitoring, analysis of food and drink safety, prevention and treatment of pathogenic infections,

studies of bacterial antibiotic resistance, in combatting biocorrosion and in biodefense (Figure 1) [1-

8]. Emergencies of these cases require robust and specific momentary analysis of trace amounts of

bacteria, at their “alarm” levels, and, thus, place very special requirements on analytical tools used.

Recent $1.7 billion Human Microbiome Project further outlined the importance of human microbiota

(a microbial community itself) and microbiome (the genetic signatures of the microbial communities)

in human health and development [9], and how changes in bacterial diversity (dysbiosis) are linked

to the progression of such diseases as diabetes, gastrointestinal diseases, colorectal and liver cancers

[10],[1]. These recent discoveries revolutionized our understanding of bacteria’s role in human health

and transformed our knowledge of disease prognosis and treatment; they also placed in focus the

necessity of new complex analytical tools for multiplex bacterial analysis. This Opinion overviews

basic concepts and last-two-years advances in electrochemical sensors for microbial analysis.

II. “Golden standard” approaches for bacterial detection and analysis.

Bacterial cell properties predetermine basic strategies for microbial analysis, which, depending on

the required information, can include analysis of whole cells, genetic or protein content isolated from

microbial cells, or products of cell activity (Figure 2A). The most conventional test is a

microbiological culture – a primary diagnostic tool that involves bacterial growth on agar plates and

further morphological and biochemical identification of bacteria and their quantification based on the

number of colonies they form on the agar plates – colony forming units (CFU). This “golden

standard” approach, however, may be insufficiently sensitive and may overlook some non-culturable

bacterial strains [11]. It takes from 24 h to several weeks of bacterial growth, this time being

unacceptable in the case of alarm or biologically critical situations. Immunological analysis of

microbes by enzyme-linked immunosorbent assays (ELISA) allows specific detection of whole

bacterial cells, however, it may suffer from insufficient sensitivity (104-105 CFU mL-1 limits of

detection, LOD [11]) and cross interference [12,13].

The polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) analyze bacteria-

isolated and amplified DNA/RNA with high specificity and sensitivity of 103-104 CFU mL-1 [11,13].

Though PCR assays dominate the market and are completely accommodated for routine biomedical

and industrial applications, PCR amplification may result in the erroneous quantification of organisms

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and inadequate sequence replication [14,15]. Non-static conditions of handling and storage of

microbial samples may also result in misleading data due to unbalanced selective growth of one

microbes over others [15], while different efficiency of DNA extraction protocols can result in 100-

fold differences in quantities of DNA extracted from different species [14]. It is also time-consuming

(up to 15 h, depending on the desired final DNA/RNA concentrations) [16].

Any technologies offering the required selectivity and sensitivity of analysis, higher speed/lower cost,

and eliminating sample amplification steps are of immediate research and market interests.

Electrochemistry with its possibility of rapid and accurate detection, low cost and power

requirements, small equipment size and easy adaptation for in-field analysis or point-of-care-testing

(POCT) is thus most suited for bacterial analysis. Currently, bacterial electroanalysis focuses on: i)

products of bacterial metabolism and cell lysates (mycotoxins are left beyond the scope of this

opinion); ii) DNA and RNA extracted from bacteria; and iii) whole bacterial cells (Figure 2B).

III. Metabolic bacterial sensors

Electrochemical monitoring of bacterial metabolism, such as differences in gas production or oxygen

consumption, is a powerful tool for detection and discrimination of live bacterial cells at both strain

and sub-species levels [17,18]. Recent approaches tend to target more specific metabolic pathways

and rely on amplifications schemes that allow accumulating the electrochemically detected product.

Redox-active pyocyanine, a water-soluble pigment and secondary metabolite produced exclusively

by Pseudomonas aeruginosa (co-cultured with other pathogenic bacteria), was voltammetrically

detected in human samples at carbon screen-printed electrodes (SPE) [19]. Whereas,

electrochemically inactive bacterial products such as N-acyl-homoserine-lactones, Gram-negative

bacteria quorum signaling molecules, could be targeted by electrochemical molecularly imprinted

polymer (MIP) sensors [20]. Electroanalysis of redox-active yet insoluble formazan produced in the

microbial suspension, 1h-incubated with a tetrazolium salt and then thermally lysed, allowed 28 CFU

mL-1 detection of viable bacterial cells [21].

S. aureus-secreted hyaluronidase was identified in wound fluids by detecting enzymatic degradation

of hyaluronic acid methacrylate coating on porous Si in a 3 h reaction [22]. Over-expression of

another bacterial enzyme - β-galactosidase - in E. coli infected by lacZ operon-engineered T7 phage

allowed 105/102 CFU mL-1 E. coli detection in aqueous samples by electrochemically detecting p-

aminophenol produced from 3 h/7 h bio-transformed 4-aminophenyl-β-galactopyranoside [23]. Lysis

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(by sonication) of 4-8 h pre-enriched E.coli and Enterococcus spp. samples enabled even more

sensitive analysis of bacteria (10 and 1 CFU mL-1, correspondingly) [24]. Here, β-galactosidase and

β-glucuronidase released from E. coli and β-glucosidase released from Enterococcus spp.

enzymatically digested their substrates and produced corresponding nitro- and amino-phenols were

voltammetrically detected at carbon SPE.

A more general but still very efficient metabolic approach explores electrocatalytic activity of

bacterial enzymes mediated by proper substrates. p-Benzoquinone as a redox mediator allowed

detecting 103-109 CFU mL-1 of E. coli and its discrimination from other bacteria, including drug-

resistant types [25]. E. coli and N. gonorrheae, captured on anti-bacterial antibody-modified gold

SPE, were selectively 106 and 107 CFU mL-1 detected through electro-enzymatic activity of their

cytochrome c oxidases in reaction with N,N,N´,N´-tetramethyl-para-phenylene-diamine as a mediator

[26]. This inexpensive assay was found suitable for assessment of antibiotic treatment procedures. A

promising modification of this approach is assessment of bacterial metabolism at

ultramicroelectrodes by analysis of electrochemical collision transients produced by cells [27].

Selectivity of bacterial detection stemmed from varying bioelectrocatalytic activity of E. coli and

Stenotrophomonas maltophilia cells oxidizing/reducing different type redox mediators with different

rates, which also allowed cell viability tests most suitable for antimicrobials screening.

IV. Electrochemical analysis of bacterial DNA and RNA

Electroanalytical schemes for bacteria-extracted DNA/RNA are general, i.e. applicable for any

DNA/RNA analysis, and just require information on genomic DNA or ribosomal rRNA sequence

composition characteristic for particular bacterial species [28-30]. Without additional

amplification/enrichment steps, bacterial analysis may be insufficient (Figure 3). Large genomic

DNA extracted from cells is low-suitable for immediate electroanalysis, and is fragmented by using

restriction enzymes digesting DNA in specific sequence positions [31]. Then the obtained fragments

are typically PCR-amplified and electroanalyzed [32]. Alternatively, genomic DNA samples can

undergo loop-mediated isothermal amplification (LAMP) in solution, and different electrochemical

reactivity of redox indicators before and after LAMP allows 30-50 min detection of DNA extracted

from 30 CFU mL-1 E. coli and 200 CFU mL-1 S. aureus [33], and 2 copies of Flavobacterium

columnare DNA [34] (Figure 3A). A solid-phase isothermal recombinase polymerase amplification

enabled 30 min detection of 105 genomic units of Salmonella [35].

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PCR-free analysis of a total RNA content extracted from ribosomes, dominated by 16S rRNA, uses

internal sample amplification. Actively grown cells can have up to 20 000 ribosomes (depending on

bacterial species), which yield a larger number of rRNA copies compared to one genomic DNA per

each bacterial cell [36-39]. In this case, electrochemical enzyme-linked assays are used as they offer

one the highest sensitivities/lowest LOD of rRNA detection (Figure 3B). Bioelectrocatalytic

amplification of DNA hybridization by horseradish peroxidase (HRP) as a label allowed detection of

8 fM E.coli´s 16S rRNA on ternary DNA-mercaptohexanol-dithiothreitol self-assembled monolayers

(SAM) [39] and improved sensing of Legionella´s 16S rRNA and DNA on thioaromatic-DNA SAMs

composed of p-aminophenol and p-mercaptobenzoic acid [37]. DNA sandwich assays on magnetic

beads (MBs), in which DNA/RNA capturing and pre-concentration on MBs and magnetic bio-

separation facilitates complex matrix analysis, offer a further analytical improvement, by lowering

LOD to 3.2 fM [38] and 1 fM 16S rRNA [36] isolated from beer-spoilage bacterium L. brevis. In both

cases, inexpensive hydrolase labels - lipase and cellulase – digested either ferrocene-labelled

synthetic ester SAM on gold [40] or insulating nitrocellulose films formed on graphite electrodes

[41], and this changed the electronic properties of the modified electrodes after their exposure to

enzyme-labelled sandwich assemblies. Overall, such enzymatic amplification schemes allow

interference-free DNA/RNA analysis comparable to PCR-based assays.

V. Whole cells analysis

The sought specificity and sensitivity of bacterial analysis is achieved by combination of the bio-

recognition abilities of aptamers, antibodies, peptides, and cell-imprinted matrices with

electrochemical methodologies. Due to the large microscopic size of bacterial cells, their binding

changes significantly electrical properties of bio-recognition interfaces, and that can be detected by a

variety of techniques; surface fouling by competitive bacterial species being one of the main

analytical issues.

Impedimetric analysis is most frequently used to detect changes in interfacial properties of bio-

modified electrodes after bacterial binding (Figure 4A). Electrochemical impedance spectroscopy

(EIS) allows 10-60 min bacterial detection at antibody- (10 CFU mL-1 E. coli [42]; 104 CFU mL-1 S.

pyogenes [43]; 100 CFU mL-1 E. coli [44];and 5.5 CFU mL-1 Listeria monocytogenes [45]), aptamer-

(600 CFU mL-1 S. eneteritidis [46]), and antimicrobial peptide (AMP)- (103 CFU mL-1 E.coli and

Salmonella [47]) modified electrodes. In the latter, AMP immobilized on gold interdigitated

microelectrodes was capable of binding to negatively-charged phospholipids of Gram-negative

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bacteria membranes, which provided a very generic platform for pathogen detection. Assays relying

on antibodies and aptamers are more specific, though, still responding to other bacterial species

[42,43,46]. Surface modifications preventing non-specific bacteria binding, such as 3D-interdigitated

electrodes separated by insulating layers [48] or surfaces blocked with poly(ethyleneglycol) [49],

slowing down discharge of the typical redox indicator - ferricyanide [50], allowed selective and 100-

10 CFU mL-1 sensitive detection of E. coli in the presence of other pathogenic bacteria. Artificial bio-

recognition sensors, such as cell-imprinted polymer sensors based on Staphylococcus epidermidis-

imprinted in electropolymerized 3-aminophenylboronic acid [51] and E.coli-imprinted ultrathin silica

films [52], were also reported as interference free, providing 103–107 CFU mL-1 and <1 CFU mL-1

bacterial detection, correspondingly.

Adaptations of nanopore technologies for bacterial cell analysis also exploit specific binding of cells

to either antibody [53] or AMP [54]: cell binding blocks nanochannels of porous membranes where

antibody/AMP immobilized in (Figure 4B). E.coli-associated blockage of nanochannels inhibited

redox indicator reaction already at 10 CFU mL-1[53], while broad recognition of outer membrane

liposaccharides by AMP made this assay generally applicable for any Gram-negative bacteria

detection [54].

Most sensitive are electrochemical cell-ELISA adaptations that use HRP and alkaline phosphatase as

bioelectrocatalytic labels [55-57], with their substrates either being electrochemically recycled

[55,56] or precipitating at electrodes [57] (Figure 4C). Assembly of an ELISA sandwich on MBs

pre-concentrates bacterial samples, and immune-magnetic separation of captured bacteria from

complex bacterial matrices increase both the selectivity and sensitivity of detection to 1.4 CFU mL-1

of Salmonella or 1 CFU mL-1 of S. aureus [56]. Redox inactive hydrolase labels such as urease [58]

or cellulase, earlier used in RNA and protein sandwich assays [36,59], also allow sensitive and

selective 12 CFU mL-1 [58] and 1 CFU mL-1 detection of E.coli with antibody/bacterium/aptamer

assemblies [60]. In both cases, products of hydrolysis were electrochemically detected: urea bio-

transformation increased the impedance of the system, while biocatalytic digestion of nitrocellulose

films on graphite electrodes increased their electronic conductivity.

Replacement of aptamers and antibodies by bacteriophages (aka phages) – chemically and thermally

stable virus nanoparticles able of specifically infecting host bacteria - allows not only sensitive and

selective bacterial analysis, but also assessment of cell viability. Peptides on the phage surface show

aptamer properties, exhibiting high affinity for bacterial surface proteins; in addition, binding affinity

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properties of phages can be modulated, both chemically and genetically. Phages can be used dually

in bacterial biosensing: as biorecongition probes capturing bacteria (Figure 4A) and as lysis agents,

destroying infected bacterial cells.

Physical adsorption of phages on electrodes results in their random orientation and, as a result,

selective analysis with not impressive LOD (103 CFU mL−1 E.coli cells) [61]. Phage orientation was

improved by its covalent directed binding combined with the alternating electric field-modulation of

phage orientation [62]. The increased the number of T4 phages properly oriented for E. coli binding

improved LOD to 100 CFU mL–1. Covalent attachment of Salmonella-specific M13 phage to

polytyramine-modified electrodes resulted in a very similar Salmonella quantification [63]. With

time, responses of such assays dropped down because of bacterial lysis. Such lysis was used for

selective 103 CFU mL-1 impedimetric detection of the E. coli B strain, after its exposure to T2 phage

covalently attached to polyethylenimine/carbon nanotubes-modified electrode under positive

potential polarization [64]. On the other hand, sensitivity of analysis can be further improved to

14 CFU mL-1 at the expense of assay time, by using non-lytic phages such as M13 phage recognizing

F+ pili of several E.coli strains [65], with this LOD not yet reaching the best results obtained with

antibodies and aptamers.

VI. Conclusions and perspectives

Many electrochemical bacterial assays overviewed here may successfully compete with existing

optical and microbiological testing approaches dominating the market in either cost or sensitivity, or

selectivity, or applicability for in-field analysis and POCT. However, despite a huge progress in

electrochemical microbial sensing and enormous market demands, commercially available solutions

are either still at the development stage or does not meet application requirements, including assay

validation, time, portability or autonomy of application. Their industrial acceptance or wider

clinical/biomedical applications are still limited, not the least, because of insufficient addressing of

the existing complex issues in analysis of pathogens and infections they cause. Already briefly

discussed, they are condensed below.

(1) The necessity of fast and reliable detection of alarm concentrations of pathogens in large-volume

samples. It poses the question on how efficiently can 1-102 CFU be pre-concentrated and detected

e.g. in 1 L samples, a problem closely connected with the problem of sample preparation/enrichment

for analysis, including processing of air and soil samples.

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(2) The necessity of fast and reliable analysis of specific bacteria in a large excess of other bacterial

species (in seawater, gut/nasal fluids etc.). Specificity for individual pathogens may still be an issue.

(3) A concomitant to (1) quick and sensitive discrimination between live and dead species at a single

cell level.

(4) Validation of analytical results for real-world sample analysis. Such validation is often absent or

does not satisfy LOD/reproducibility requirements; there are many artefacts still reported in literature.

(5) The necessity of continuous/autonomous monitoring systems (for water, food or biocorrosion

control) and of POCT and in-field testing systems to be operated by minimally trained personal.

While electrochemistry offers attractive solutions for inexpensive yet efficient POCT and in-field

testing systems for bacterial analysis, other issues are still to be resolved in complex biosensor

platforms that to be developed in close collaboration with microbiologists, medical doctors, engineers

and analytical chemists. To achieve that, most perspective, in my opinion, are immunomagnetic and

phage-assisted assays for whole cell analysis and recent approaches to electrochemical sensing for

viable bacteria metabolism. Adapted for lab-on-chip or out-of-lab/POCT formats, they could enhance

both diagnostic and therapeutic capacities of the society, combatting infectious and dysbiosis-

triggered diseases; specific out-breaks of new pathogens, and the spread of antimicrobial resistance,

with this addressing the Universal Health Coverage principles meaning everyone’s access to the

health services they need, timely and at affordable price [2].

Acknowledgements

This work was done within the H2O2-MSCA-ITN-2018 “Break Biofilms” Training Network

project, grant agreement 813439.

Declaration of interest: none

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Figure captions

Figure 1. Summary of the application fields strongly requiring efficient microbial biosensors

(information adapted from references [1-8]).

Figure 2. (A) Schematic representation of the bacterial cell. Bacteria are prokaryotes and as such,

they miss organelles such as mitochondria or chloroplasts, and possess instead ribosomes, which

number can reach several dozen thousands. In contrast to eukaryotes, bacterial genetic information is

stored in cytoplasm, in the form of DNA loops, but not in the nucleus, and several plasmids. Based

on their wall structure/ability of staining all bacteria are divided into Gram-positive (can be stained

with crystal violet) and Gram-negative species (cannot be stained). The cell envelope of Gram-

negative bacteria is composed of a peptidoglycan cell wall sandwiched between the plasma membrane

(a phospholipid bilayer) and the outer membrane (phospholipids/liposaccharides impregnated with

proteins), with both cell stiffness and strength resulting from the outer membrane properties [66]. The

cell envelop of Gram-positive species is composed of several peptidoglycan layers and is much

thicker than that of Gram-negative species. (B) Schematic representation of general electrochemical

approaches for bacterial electroanalysis including electroanalysis of (i) products of bacterial

metabolism or cell lysates; (ii) DNA and RNA extracted from bacteria; and (iii) whole bacterial cells

at bare and modified electrodes. Electrode modification may include (ii) DNA probes or (iii)

antibodies, while a typical redox indicator is ferricyanide present in solution (O/R).

Figure 3. (A) Schematic representation of PCR- and LAMP-amplified assays for bacterial DNA,

including steps of DNA extraction, amplification and either detection with electronic redox-labeled

hairpin beacons (green ovals: methylene blue redox labels covalently attached to the free end of the

hairpin sequence) [32] or electrochemical detection of redox molecules (red circles: methylene blue

or other electroactive DNA-intercalating species) more/less available for electrode reactions

before/after they intercalate into solution-present (not immobilized at electrodes) bacterial DNA

continuously amplified by LAMP [34]. (B) Electrochemical DNA sandwich assays, on DNA-probe

modified solid electrodes and on magnetics beads, with either a redox enzyme (horseradish

peroxidase or alkaline phosphatase) [39] or such hydrolase as cellulase as labels [36]. Bacterial DNA

14

or RNA is captured by the DNA probe and after that reacts with the biotinylated reporter DNA

consequently labelled with the enzyme through the streptavidin-biotin linkage (shown in blue-red).

Bioelectrocatalytic amplification of the signal from the redox enzyme is provided by electrochemical

recycling of the redox mediator M (e.g. catechol species) operating as a second substrate for the

enzyme. Cellulase, in its turn, enzymatically digests its substrate nitrocellulose film on the electrode

surface (shown in blue), which results in changes of the electrical properties of the film that can be

detected both with and without the redox indicator such as ferricyanide (O/R).

Figure 4. Schematic representations of some examples of bacterial cell assays. (A) With the antibody-

aptamer-, peptide-, phage- and cell-imprinted polymer-modified electrodes. Binding of bacteria to

the bio-modified electrodes increases impedance in these systems and slows down the

electrochemical reaction of a redox indicator, typically, ferricyanide (O/R); (B) In nanochannels,

where bacterial cell binding to antibody- or peptide-modified nanochannels blocks them for the redox

indicator reaction [53] (with either ferrocene or ferricyanide). (C) Electrochemical whole-cell ELISA

adapted to magnetic beads [56] (mechanistically similar to Figure 3B).

15

Figures

Figure 1

16

Figure 2

17

Figure 3

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Figure 4