Review—Electrochemical Approaches and Advances towards the Detection of Drug Resistance

In order to study drug resistance electrochemically, the redox properties of drug compounds must be well understood. Based on their chemical structure, FDA approved antibiotics are commonly categorized into eight different classes, as summarized in Table I.^16–19 Since 2009, electrochemical investigations focused mostly on β-lactams,^20–28 aminoglycoside,^29,30 quinolones^{10–13,31–43} and macrolides^44–50 due to their effectiveness against a large number of bacterial diseases. In the following, we discuss some of the most commonly prescribed antibiotics and their electrochemical properties to evaluate their usefulness for electrochemical live cell studies.

Table I.  Classes of antibiotics reported in literature.^16–19

Class Name	Structure	Mechanism of Action
β-Lactam		Inhibit bacterial cell wall biosynthesis
Aminoglycosides		Inhibit bacterial protein synthesis leading to cell death
Glycopeptides		Inhibit bacterial cell wall biosynthesis
Quinolones		Inhibit bacterial DNA replication
Oxazolidinones		Inhibit bacterial protein synthesis
Tetracyclines		Inhibit bacterial protein synthesis
Macrolides		Inhibit bacterial protein synthesis
Lipopeptides		Disrupt bacterial cell membrane

Amoxicillin. The antibiotic class of β-lactams is widely used to prevent and treat various bacterial infections in humans and the field of agriculture.²³ Amoxicillin is a member of this class^20,22 and is the p-hydroxy analogue of ampicillin. It contains variable side chains, which are responsible for its rapid absorption by the human body. The electrochemistry of amoxicillin was investigated by the author Ağin in 2016²⁴ using glassy carbon electrodes (GCEs), which were modified by electropolymerization of acridine orange. The electrochemical behavior of amoxicillin was characterized by cyclic voltammetry (CV), revealing an irreversible oxidation peak at 0.92 V vs Ag/AgCl at a scan rate of 100 mV s⁻¹. This study suggests that the pH of the analyte solution has a profound effect on the peak current and potential. An optimal pH of 5.0 was reported for the determination of amoxicillin and a linear relationship between the anodic peak current as a function of the square root of the scan rate indicated a diffusion controlled process. In this study, differential pulse voltammetry (DPV) and square wave voltammetry (SWV) revealed a limit of detection (LOD) of 1.87 × 10⁻⁹ M and 1.55 × 10⁻⁸ M, respectively. No interferences were reported in this publication.

Ampicillin. An interesting strategy to detect ampicillin has been proposed by Wang et al. using aptamer based differential pulse voltammetry.²³ In this approach, a GCE was modified with double stranded DNA (dsDNA) containing an ampicillin aptamer sequence (Fig. 1a). Differential pulse voltammograms showed a pronounced reduction peak. This method resulted in an impressive detection limit of 3.2 × 10⁻¹¹ M and ampicillin was detected in real samples of milk and water.

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Ciprofloxacin (CIP). Belonging to the second generation of fluoroquinolones, CIP is used worldwide as an antimicrobial agent to treat infections in humans and livestock.^39,40 Some antibiotics, such as CIP, can react with metal ions to form stable complexes. Several studies have been conducted to electrochemically characterize CIP.^{10–13,37–40} Its detection through the complexation of CIP with Cd²⁺ was reported by Jun et al.⁴⁰ A well-defined electrochemical signal was recorded at graphene modified GCEs as a result of the complexation of Cd²⁺ by CIP. The concentration of CIP was found inversely proportional to the electrochemical signal and an irreversible oxidation peak of CIP in various electrolyte solutions, including acetate buffer solution (ABS), phosphate buffer saline (PBS) and sulphuric acid, was observed. GCE modifications using graphene resulted in a significantly enhanced anodic peak in the presence of Cd²⁺ ions. Overall, this method showed good reproducibility and high selectivity with a detection limit of 5.9 × 10⁻⁸ M. The main advantage of this approach is that the electrode did not suffer from electrode fouling which is commonly observed during the direct determination of CIP at bare GCEs. This method successfully detected CIP rapidly and sensitively in pharmaceuticals and urine samples.

Norfloxacin (NFX). This fluoroquinolone is commonly used to treat gastrointestinal, urinary and respiratory tract infections.³⁶ As shown by Ke-Jing et al., NFX was detected at multi-wall carbon nanotube (MWCNT)/nafion film-coated GCEs during voltammetry.³⁶ NFX was determined by linear sweep voltammetry at a potential range of 0.3 to 1.4 V vs Ag/AgCl, which resulted in a well-defined irreversible oxidation peak current at a peak potential of 1.11 V vs Ag/AgCl. The relationship between the peak current and the scan rate at a range of 10–250 mV s⁻¹ demonstrated that mass transfer occurs through an adsorption controlled process and that the pH of the supporting electrolyte solution has an important influence on the electrochemical reaction. The highest oxidation peak was recorded in HAc–NH₄Ac buffer solution at a pH value of 4.4. This method has been used in pharmaceuticals and urine samples with an excellent detection limit of 5 × 10⁻⁸ M. Only minimal fouling effects were observed during this procedure.

Tobramycin. Tobramycin (TOB) is an aminoglycoside, which is effective against both Gram-positive and Gram-negative bacterial infections.²⁹ A modified GCE was prepared by electropolymerization of pyrrole (Py) in the presence of tobramycin to form a molecularly imprinted polymer film on the GCE surface (PPy/GCE). The general approach of fabricating cavities in a polymer matrix based on a specific template molecule is known as Molecularly Imprinted Polymer (MIP) technique and has found useful application in a variety of research fields including electrochemistry, analytical chemistry and biology.^52–54 In the work of Gupta et al., mass transfer occurs through a diffusion controlled process where TOB interacts with pyrrole and results in an oxidation peak at 0.72 V vs Ag/AgCl. Modified TOB imprinted PPy/GCEs were electrochemically characterized by cyclic voltammetry in the presence of potassium ferricyanide in KCl and compared to a bare GCE. The reversible redox peak of potassium ferricyanide, which is observed on the bare GCE, is not shown on the modified GCE due to the presence of the polymer. Square wave voltammetry was performed revealing a favorable detection limit of 1.4 × 10⁻¹⁰ M.²⁹

Neomycin. Another example for the electrochemical characterization of aminoglycosides is neomycin, which has gained considerable attention due to its effectiveness against Gram-negative and Gram-positive bacteria to treat gastrointestinal infections and mastitis in livestock animals.³⁰ Neomycin exhibits a well-defined reduction peak at a potential of −0.2 V vs Ag/AgCl as shown by Hamnca et al. Square wave voltammetry was carried out at the surface of a polyamic acid/graphene oxide (GO)/screen-printed carbon electrode (SPCE) for the electrochemical detection of neomycin in aqueous solutions. The reduction peak current was shown to increase with increasing concentration of neomycin until the catalytic current enhancement effect at the electrode surface reached a saturation level. The limit of detection was found to be 1.07 × 10⁻⁶ M. This approach was applied to detect neomycin in urine samples, which presents a reliable and rapid alternative to common chromatography techniques.³⁰

Azithromycin. An interesting molecule of the macrolides class is azithromycin (AZM), which is effective against Gram-negative and Gram-positive bacterial infections.^45,49 AZM is highly effective against upper and lower respiratory acute tract infections such as bronchitis, pneumonia, sinusitis, pharyngitis, tonsillitis and otitis, sexually transmitted diseases, skin and soft tissue infections.^45,48,49 The electrochemical behavior of AZM has been investigated by cyclic voltammetry on MIP modified carbon paste (CP) electrodes (MIP/CPs) and non-molecularly imprinted polymer (NIP) modified CP electrode (NIP/CPs). Voltammograms show an irreversible oxidation peak at 0.8 V vs Ag/AgCl due to the oxidation of one dimethyl-amino group on the desosamine sugar part of the molecule. This work gives the lowest detection limit of 2.3 × 10⁻¹¹ M, which compares favorable over any other voltammetry methods.⁴⁴

Other existing electrochemical studies on antibiotics not discussed in detail, are summarized in Table II.

Anti-cancer drugs that were electrochemically characterized in the last decade are categorized into four groups: alkylating agents, antimetabolites, cytotoxic antibiotics, and inhibitors. While alkylating agents, such as cisplatin lead to cell death in cancer by binding to DNA, antimetabolites, such as 6-mercaptopurine and 5-fluorouracil, mimic metabolites to interfere with processes required for cell division.^58,59 Cytotoxic antibiotics, such as doxorubicin are drugs, which are used to treat cancer in addition to their antimicrobial properties.^58,59 The presented group of inhibitors consists of chemotherapeutic drugs able to inhibit various enzymes. For example, etoposide can interfere with the enzyme topoisomerase II, which is involved in relieving stress on DNA, induced by supercoiling during cell division.⁶⁰ The following section discusses recent studies on these four anti-cancer drug categories.

6-mercaptopurine (6-MP). Antimetabolite drugs have been studied extensively using electrochemical methods in the past decade, due to their common use in treating various types of cancers.^51,61–77 6-MP is an anti-cancer drug used to treat leukemia.⁷⁶ There have been several attempts to electrochemically detect 6-MP,^68,72,76 such as the recent publication of Kumar et al.,⁶⁸ who developed a method that reaches a detection limit of 7.2 × 10⁻¹⁰ M in blood plasma. In this work, the authors synthesize nitrogen-doped hollow carbon nanospheres, coated with Palladium (N-HCNS-Pd) and MIPs. These modifications help to improve the effective area and roughness factor of the electrode, which increases its sensitivity. The modified hollow carbon nanospheres are held on an ionic liquid (IL) modified pencil graphite electrode ( PGE) via electrostatic interaction. The authors utilize differential pulse anodic stripping voltammetry (DPASV) to detect 6-MP. A diffusion controlled and irreversible process for the 6-MP oxidation is presented, which involves two protons and two electrons. Notably, these electrode modifications are stable for 3 weeks if stored at room temperature and in dark conditions.⁶⁸

Other electrode modifications, such as using biomolecules are less stable, but equally valuable due to their effectiveness, as shown by Pattar et al. in 2015. In this manuscript the authors detect 5-fluorouracil (5-FU), an antimetabolite drug used to treat various cancer kinds, such as colon cancer,⁶² stomach cancer,⁶² breast cancer,⁶² and cervical cancer.⁶² Pattar et al. employ chitosan, a chemically processed form of chitin^64,65,67,75 in combination with reduced graphene oxide to modify GCEs. This combination creates a rougher electrode surface and results in an electrocatalytic effect to achieve low detection limits. By applying staircase voltammetry (SCV), an impressive LOD of 1.24 × 10⁻⁹ M was achieved.⁶⁴

Other successful electrode modification strategies involve the use of nanoparticles,^61,66 ionic liquids,⁶³ graphene oxide and carbon-nanotubes,⁷¹ methylene blue (MTB),⁶⁷ and the use of copper electrodes.⁷⁷ Important to point out is the work by Hadi et al. in 2017, utilizing a combination of graphene oxide and carbon nanotubes to modify GCEs and SPCEs. This method shows the widest analytical dynamic linear range from 0.05 to 1200 × 10⁻⁶ M, which is shown useful for the determination of 5-FU in human plasma.⁷¹

Gemcitabine (GMB) is an anti-cancer drug which is classified as an antimetabolite. Electrochemical detection of GMB can be achieved through the use of bare gold electrodes (AuE), as shown by the authors Naik et al. in 2013.⁷³ Detection of drugs at bare electrodes is commonly favorable, because it saves time and costs, but often times results in lower detection limits. Complex electrode modifications to detect GMB using a microporous metal organic framework (MMOF) (MMOF/AuNP/Au electrode) has been proposed by Florea et al. in 2015.⁵¹ These authors construct a molecularly imprinted MMOF by electropolymerizing p-aminothophenol (PATP) equipped with gold nanoparticles (AuNP) on a gold electrode surface in the presence of GMB (Fig. 1b). GMB molecules act directly as templates to create complementary pores in the framework. After the electropolymerization, GMB is extracted, leaving empty binding sites. To analyze samples, the modified electrode is immersed in a solution with GMB, which will bind to complementary sites in the network. This binding will enhance the reduction peak of an electrochemical probe (ferrocyanide/ ferricyanide) and thus, GMB is detected indirectly. This method results in a LOD of 3.4 × 10⁻¹⁵ M. This method provides not only high specificity but also high sensitivity compared to the detection at bare electrodes.⁵¹

Cytotoxic antibiotics are antibiotics that are found to have anti-tumor activities. These antibiotics have many mechanisms of action. The subgroup of anthracyclines has been studied in the literature and contains antibiotics, doxorubicin^78,79 and daunorubicin,⁸⁰ extracted from Streptomyces bacteria. Doxorubicin (DOX) was detected using voltammetry⁸¹ as well as electrochemical impedance spectroscopy (EIS).^78,79 Rezaei et al. developed two different methods to utilize immunosensor and EIS to detect doxorubicin at very low concentrations. While one method uses gold electrodes modified with thiol base sol-gel (TBSol-Gel), gold nanoparticles (AuNP), and monoclonal antibody (MAb) (MAb/AuNP/TBSol-Gel/AuE),⁷⁹ the other method modifies a stainless steel electrode (SSE) with 3-aminopropyltriethoxysilane (APTES), AuNP, and Mab (MAb/AuNP/APTES/SSE).⁷⁸ Both strategies follow a similar detection principle. Firstly, an electrode is coated with an insulating layer (TBSol-Gel or APTES) at the electrode surface. Secondly, AuNPs are deposited on the electrode by immersing the electrode in HAuCl₄ solution for 180 s at −200 mV (with Ag/AgCl as reference electrode). After that, the newly modified electrode is incubated in a doxorubicin MAb. Finally, the electrode is immersed in a 1% bovine serum albumin (BSA) to block non-specific active site. The modified electrode is immersed in a standard solution of DOX prior to sample measurements. The charge transfer resistance, which is obtained from Nyquist diagrams, is used as a signal. The MAb/AuNP/TBSol-Gel/Au and the MAb/AuNP/APTES/SS electrode result in an LOD of 1.7 × 10⁻¹³ M and 3.1 × 10⁻¹² M, respectively.

Other cytotoxic antibiotics that were studied in the past decade include epirubicin,⁸² daunorubicin,⁸⁰ and valrubicin.⁸³ A common theme in these three studies is the combination of carbon nanotubes with nanoparticles and other components to detect drugs. Shams et al.⁸² introduce a GCE modified with silver decorated MWCNT composite (Ag-MWCNT/GCE). By using square wave voltammetry, this work detects epirubicin with a LOD of 1.0 × 10⁻⁹ M. Kong et al. 2019⁸⁰ prepared a nanocomposite from nitrogen decorated reduced graphene oxide and single-walled carbon nanotubes. This nanocomposite is loaded with platinum nanoparticles (PtNP) and is then used to modify a GCE (N-rGO-SWCNTs-Pt/GCE). At optimized conditions, this method achieved an LOD of 5.7 × 10⁻⁹ M for daunorubicin. In 2015, Hajian et al.⁸³ fabricated an electrochemical sensor based on gold electrode modified with gold nanoparticles/ethylenediamine (EDA)/multi-walled carbon nanotubes (AuNPs/EDA/MWCNTs/AuE). This sensor can detect valrubicin with an LOD of 1.8 × 10⁻⁸ M in citrate buffer (pH 4.0) and 0.1 M KCl.

Alkylating agents include temodal,⁸⁴ ifosfamide,⁸⁵ and cisplatin.^86,87 Temodal was recently detected by the authors Jahandari et al. in 2019, who propose the use of gold nanoparticles (AuNP) and dsDNA to modify a pencil graphite electrode.⁸⁴ One unique feature of this work is the use of computational chemistry to study the intercalations between temodal and guanine in dsDNA structure, which is used to detect the drug. In particular, this interaction between drug and dsDNA decreases the current of the oxidation peak of the guanine base. This method achieved an LOD of 1.0 × 10⁻⁹ M.

Other chemotherapeutic drugs that have been electrochemical studied in the past, but are not reviewed in detail here are etoposide⁸⁵ and the tyrosine kinase inhibitor erlotinib.⁷⁴ A summary of anti-cancer drugs investigated by electrochemistry is presented in Table III.