electrochemical assays for microbial analysis: how …...iii. metabolic bacterial sensors...
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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: [email protected]
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
References
1. Carding S, Verbeke K, Vipond DT, Corfe BM, Owen LJ: Dysbiosis of the gut microbiota in
disease. Microb. Ecol. Health Disease 2015, 26:26191.
2. Dye C: After 2015: infectious diseases in a new era of health and development. Philos. Trans.
Royal Soc. B: Biol. Sci. 2014, 369:20130426.
3. Jansen HJ, Breeveld FJ, Stijnis C, Grobusch MP: Biological warfare, bioterrorism, and
biocrime. Clin. Microbiol. Infect. 2014, 20:488-496.
4. Koch GH, Brongers MP, Thompson NG, Virmani YP, Payer JH: Cost of corrosion in the
United States. In Handbook of environmental degradation of materials. 2005:3-24.
5. Moiseeva L, Kondrova O: Biocorrosion of Oil and Gas Field Equipment and Chemical
Methods for Its Suppression. I. Protect. Metals 2005, 41:385-393.
9
6. Velusamy V, Arshak K, Korostynska O, Oliwa K, Adley C: An overview of foodborne
pathogen detection: In the perspective of biosensors. Biotechnol. Adv. 2010, 28:232-254.
7. WHO: New report calls for urgent action to avert antimicrobial resistance crisis.
https://www.healthpolicy-watch.org/no-time-to-wait-amr-could-cause-10-million-deaths-
annually-by-2050-warns-un-report/. Accessed 29.12.2019.
8. WHO: https://www.who.int/water_sanitation_health/publications/jmp-report-2019/en/;
https://www.who.int/water_sanitation_health/diseases-risks/en/. Accessed 29.12.2019.
9. Proctor L, LoTempio J, Marquitz A, Daschner P, Xi D, Flores R, Brown L, Ranallo R, Maruvada
P, Regan K, et al.: A review of 10 years of human microbiome research activities at the
US National Institutes of Health, Fiscal Years 2007-2016. Microbiome 2019, 7:31.
10. Proctor LM, Creasy HH, Fettweis JM, Lloyd-Price J, Mahurkar A, Zhou W, Buck GA, Snyder
MP, Strauss JF, Weinstock GM, et al.: The Integrative Human Microbiome Project.
Nature 2019, 569:641-648.
11. Wang Y, Salazar JK: Culture-independent rapid detection methods for bacterial pathogens
and toxins in food matrices. Compr. Rev. Food Sci. Food Saf. 2016, 15:183-205.
12. Verma J, Saxena S, Babu SG: ELISA-based identification and detection of microbes. In
Analyzing Microbes. Springer Protocols Handbooks. Edited by Arora D, Da sS, Sukumar
M: Springer, Berlin, Heidelberg; 2013.
13. Law JW-F, Ab Mutalib N-S, Chan K-G, Lee L-H: Rapid methods for the detection of
foodborne bacterial pathogens: principles, applications, advantages and limitations.
Front. Microbiol. 2015, 5.
14. Costea PI, Zeller G, Sunagawa S, Pelletier E, Alberti A, Levenez F, Tramontano M, Driessen
M, Hercog R, Jung F-E, et al.: Towards standards for human fecal sample processing in
metagenomic studies. Nature Biotechnol. 2017, 35:1069.
15. McDonald D, Hyde E, Debelius JW, Morton JT, Gonzalez A, Ackermann G, Aksenov AA,
Behsaz B, Brennan C, Chen Y, et al.: American gut: an open platform for citizen science
microbiome research. mSystems 2018, 3:e00031-00018.
16. Nolan T, Hands RE, Bustin SA: Quantification of mRNA using real time RT-PCR. Nature
Protocols 2006, 1:1559-1582.
17. Karasinski J, Andreescu S, Sadik OA, Lavine B, Vora MN: Multiarray sensors with pattern
recognition for the detection, classification, and differentiation of bacteria at
subspecies and strain levels. Anal. Chem. 2005, 77:7941-7949.
18. Pavlou AK, Magan N, Jones JM, Brown J, Klatser P, Turner APF: Detection of
Mycobacterium tuberculosis (TB) in vitro and in situ using an electronic nose in
combination with a neural network system. Biosens. Bioelectron. 2004, 20:538-544.
19. Santiveri CR, Sismaet HJ, Kimani M, Goluch ED: Electrochemical detection of Pseudomonas
aeruginosa in polymicrobial environments. ChemistrySelect 2018, 3:2926-2930.
20. Jiang H, Jiang D, Shao J, Sun X: Magnetic molecularly imprinted polymer nanoparticles
based electrochemical sensor for the measurement of Gram-negative bacterial quorum
signaling molecules (N-acyl-homoserine-lactones). Biosens. Bioelectron. 2016, 75:411-
419.
21. Ishiki K, Nguyen DQ, Morishita A, Shiigi H, Nagaoka T: Electrochemical detection of viable
bacterial cells using a tetrazolium salt. Anal. Chem. 2018, 90:10903-10909.
22. Tücking K-S, Vasani RB, Cavallaro AA, Voelcker NH, Schönherr H, Prieto-Simon B: Porous
silicon: hyaluronic acid–modified porous silicon films for the electrochemical sensing
of bacterial hyaluronidase. Macromol. Rapid Commun. 2018, 39:1870044.
23. Wang D, Chen J, Nugen SR: Electrochemical detection of Escherichia coli from aqueous
samples using engineered phages. Anal. Chem. 2017, 89:1650-1657.
10
24. Adkins JA, Boehle K, Friend C, Chamberlain B, Bisha B, Henry CS: Colorimetric and
electrochemical bacteria detection using printed paper- and transparency-based
analytic devices. Anal. Chem. 2017, 89:3613-3621.
*Ultrasensitive and simple assay for pre-enriched bacterial samples applicable for practical in-field
detection of food and waterborne bacteria.
25. Sun J, Warden AR, Huang J, Wang W, Ding X: Colorimetric and electrochemical detection
of Escherichia coli and antibiotic resistance based on a p-benzoquinone-mediated
bioassay. Anal. Chem. 2019, 91:7524-7530.
26. Kuss S, Couto RAS, Evans RM, Lavender H, Tang CC, Compton RG: Versatile
electrochemical sensing platform for bacteria. Anal. Chem. 2019, 91:4317-4322.
27. Gao G, Wang D, Brocenschi R, Zhi J, Mirkin MV: Toward the detection and identification of
single bacteria by electrochemical collision technique. Anal. Chem. 2018, 90:12123-
12130.
**Novel electrochemical approach for electroanalysis of specific redox activities of bacterial cells
and cell viability, prospective for rapid screening of antmicrobila agents.
28. Campuzano S, Yáñez-Sedeño P, Pingarrón JM: Electrochemical biosensing for the diagnosis
of viral infections and tropical diseases. ChemElectroChem 2017, 4:753-777.
29. Yu HLL, Maslova A, Hsing IM: Rational design of electrochemical DNA biosensors for
point-of-care applications. ChemElectroChem 2017, 4:795-805.
30. Ferapontova E: Basic concepts and recent advances in electrochemical analysis of nucleic
acids. Curr. Opin. Electrochem. 2017, 5:218-225.
31. Falcone N, She Z, Chen C, Dong B, Yi D, Kraatz H-B: Demonstration of a tailorable and
PCR-free detection of Enterococcus DNA isolated from soil samples. Anal. Methods
2017, 9:1643-1649.
*A rare example of practical environmental analysis of bacterial DNA extracted from bacterial soil
samples.
32. Ferguson BS, Buchsbaum SF, Swensen JS, Hsieh K, Lou X, Soh HT: Integrated microfluidic
electrochemical DNA sensor. Anal. Chem. 2009, 81:6503-6508.
33. Safavieh M, Ahmed MU, Ng A, Zourob M: High-throughput real-time electrochemical
monitoring of LAMP for pathogenic bacteria detection. Biosens. Bioelectron. 2014,
58:101-106.
34. Martin A, Grant KB, Stressmann F, Ghigo J-M, Marchal D, Limoges B: Ultimate single-copy
DNA detection using real-time electrochemical LAMP. ACS Sensors 2016, 1:904-912.
35. Sánchez-Salcedo R, Miranda-Castro R, de los Santos-Álvarez N, Lobo-Castañón MJ: On-gold
Recombinase Polymerase Primer elongation for electrochemical detection of bacterial
genome: mechanism insights and influencing factors. ChemElectroChem 2019, 6:793-
800.
36. Fapyane D, Nielsen JS, Ferapontova EE: Electrochemical enzyme-linked sandwich assay
with a cellulase label for ultrasensitive analysis of synthetic DNA and cell-isolated
RNA. ACS Sensors 2018, 3:2104-2111.
*Robust and inexpensive electrochemical technology for ultrasensitive analysis of unamplified
bacterial DNA and RNA samples with an inexpensive hydrolase label.
37. Miranda-Castro R, Sánchez-Salcedo R, Suárez-Álvarez B, de-los-Santos-Álvarez N, Miranda-
Ordieres AJ, Jesús Lobo-Castañón M: Thioaromatic DNA monolayers for target-
amplification-free electrochemical sensing of environmental pathogenic bacteria.
Biosens. Bioelectron. 2017, 92:162-170.
*Simple and efficient strategy of tuning properties of DNA-sensing SAM to improve their stability
and sensitivity of rRNA assaying.
11
38. Shipovskov S, Saunders AM, Nielsen JS, Hansen MH, Gothelf KV, Ferapontova EE:
Electrochemical sandwich assay for attomole analysis of DNA and RNA from beer
spoilage bacteria Lactobacillus brevis. Biosens. Bioelectron. 2012, 37:99-106.
39. Wu J, Campuzano S, Halford C, Haake DA, Wang J: Ternary surface mono layers for
ultrasensitive (zeptomole) amperometric detection of nucleic acid hybridization
without signal amplification Anal. Chem. 2010, 82:8830-8837.
40. Ferapontova EE, Hansen MN, Saunders AM, Shipovskov S, Sutherland DS, Gothelf KV:
Electrochemical DNA sandwich assay with a lipase label for attomole detection of
DNA. Chem. Commun. 2010, 46:1836-1838.
41. Fapyane D, Ferapontova EE: Electrochemical assay for a total cellulase activity with
improved sensitivity. Anal. Chem. 2017, 89:3959-3965.
42. Maalouf R, Fournier-Wirth C, Coste J, Chebib H, Saïkali Y, Vittori O, Errachid A, Cloarec J-P,
Martelet C, Jaffrezic-Renault N: Label-free detection of bacteria by electrochemical
impedance spectroscopy: comparison to surface plasmon resonance. Anal. Chem. 2007,
79:4879-4886.
43. Ahmed A, Rushworth JV, Wright JD, Millner PA: Novel impedimetric immunosensor for
detection of pathogenic bacteria Streptococcus pyogenes in human saliva. Anal. Chem.
2013, 85:12118-12125.
44. Li Z, Fu Y, Fang W, Li Y: Electrochemical impedance immunosensor based on self-
assembled monolayers for rapid detection of Escherichia coli O157:H7 with signal
amplification using lectin Sensors 2015, 15:19212-19224.
45. Chiriacò MS, Parlangeli I, Sirsi F, Poltronieri P, Primiceri E: Impedance sensing platform for
detection of the food pathogen Listeria monocytogenes. Electronics 2018, 7:347-.
46. Labib M, Zamay AS, Kolovskaya OS, Reshetneva IT, Zamay GS, Kibbee RJ, Sattar SA, Zamay
TN, Berezovski MV: Aptamer-based impedimetric sensor for bacterial typing. Anal.
Chem. 2012, 84:8114-8117.
47. Mannoor MS, Zhang S, Link AJ, McAlpine MC: Electrical detection of pathogenic bacteria
via immobilized antimicrobial peptides. Proc. Natl. Acad. Sci. USA 2010, 107:19207-
19212.
48. Brosel-Oliu S, Ferreira R, Uria N, Abramova N, Gargallo R, Muñoz-Pascual F-X, Bratov A:
Novel impedimetric aptasensor for label-free detection of Escherichia coli O157:H7.
Sens. Actuat. B: Chem. 2018, 255:2988-2995.
49. Jijie R, Kahlouche K, Barras A, Yamakawa N, Bouckaert J, Gharbi T, Szunerits S, Boukherroub
R: Reduced graphene oxide/polyethylenimine based immunosensor for the selective
and sensitive electrochemical detection of uropathogenic Escherichia coli. Sens. Actuat.
B: Chem. 2018, 260:255-263.
50. Salimian R, Kékedy-Nagy L, Ferapontova EE: Specific picomolar detection of a breast
cancer biomarker HER-2/neu protein in serum: electrocatalytically amplified
electroanalysis by the aptamer/PEG-modified electrode. ChemElectroChem 2017, 4:872-
879.
51. Golabi M, Kuralay F, Jager EWH, Beni V, Turner APF: Electrochemical bacterial detection
using poly(3-aminophenylboronic acid)-based imprinted polymer. Biosens. Bioelectron.
2017, 93:87-93.
52. Jafari H, Amiri M, Abdi E, Navid SL, Bouckaert J, Jijie R, Boukherroub R, Szunerits S:
Entrapment of uropathogenic E. coli cells into ultra-thin sol-gel matrices on gold thin
films: A low cost alternative for impedimetric bacteria sensing. Biosens. Bioelectron.
2019, 124-125:161-166.
12
**Excellent example of ultrasensitive bacterial detection with an artificial bireceptor-based cell-
imprinted polymer sensor.
53. Cheng MS, Lau SH, Chow VT, Toh C-S: Membrane-based electrochemical nanobiosensor
for Escherichia coli detection and analysis of cells viability. Environ. Sci. Technol. 2011,
45:6453-6459.
54. Reta N, Michelmore A, Saint CP, Prieto-Simon B, Voelcker NH: Label-free bacterial toxin
detection in water supplies using porous silicon nanochannel sensors. ACS Sensors
2019, 4:1515-1523.
** Simple and efficient nanopore (nanochannel) technology for Gram-negative bacteria detection.
55. Campuzano S, de Ávila BE-F, Yuste J, Pedrero M, García JL, García P, García E, Pingarrón
JM: Disposable amperometric magnetoimmunosensors for the specific detection of
Streptococcus pneumoniae. Biosens. Bioelectron. 2010, 26:1225-1230.
56. Esteban-Fernández de Ávila B, Pedrero M, Campuzano S, Escamilla-Gómez V, Pingarrón JM:
Sensitive and rapid amperometric magnetoimmunosensor for the determination of
Staphylococcus aureus. Anal. Bioanal. Chem. 2012, 403:917-925.
57. Ruan C, Yang L, Li Y: Immunobiosensor chips for detection of Escherichia coli O157:H7
using electrochemical impedance spectroscopy. Anal. Chem. 2002, 74:4814-4820.
58. Yao L, Wang L, Huang F, Cai G, Xi X, Lin J: A microfluidic impedance biosensor based on
immunomagnetic separation and urease catalysis for continuous-flow detection of E.
coli O157:H7. Sens. Actuat. B: Chem. 2018, 259:1013-1021.
**A highly efficient ELISA-on-magnetic-beads assay with a non-traditional urease label operating
in the continuos flow mode for sensitive bacterial analysis.
59. Malecka K, Pankratov D, Ferapontova EE: Femtomolar electroanalysis of a breast cancer
biomarker HER-2/neu protein in human serum by the cellulase-linked sandwich assay
on magnetic beads. Anal. Chim. Acta 2019, 1077:140-149.
60. Pankratov D, Bendixen M, Gosewinkel U, Ferapontova E: Cellulase-linked immunomagnetic
analysis of microbes: specific and sensitive single-cell detection. submitted 2019.
61. Moghtader F, Congur G, Zareie HM, Erdem A, Piskin E: Impedimetric detection of
pathogenic bacteria with bacteriophages using gold nanorod deposited graphite
electrodes. RSC Adv. 2016, 6:97832-97839.
62. Richter Ł, Bielec K, Leśniewski A, Łoś M, Paczesny J, Hołyst R: Dense layer of
bacteriophages ordered in alternating electric field and immobilized by surface
chemical modification as sensing element for bacteria detection. ACS Appl.Mater.
Interfaces 2017, 9:19622-19629.
63. Niyomdecha S, Limbut W, Numnuam A, Kanatharana P, Charlermroj R, Karoonuthaisiri N,
Thavarungkul P: Phage-based capacitive biosensor for Salmonella detection. Talanta
2018, 188:658-664.
64. Zhou Y, Marar A, Kner P, Ramasamy RP: Charge-directed immobilization of bacteriophage
on nanostructured electrode for whole-cell electrochemical biosensors. Anal. Chem.
2017, 89:5734-5741.
65. Sedki M, Chen X, Chen C, Ge X, Mulchandani A: Non-lytic M13 phage-based highly
sensitive impedimetric cytosensor for detection of coliforms. Biosens. Bioelectron. 2020,
148:111794.
*A new strategy of using engineered non-lytic phages for electrochemical biosensing of bacterial
cells.
66. Rojas ER, Billings G, Odermatt PD, Auer GK, Zhu L, Miguel A, Chang F, Weibel DB, Theriot
JA, Huang KC: The outer membrane is an essential load-bearing element in Gram-
negative bacteria. Nature 2018, 559:617-621.
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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