In this section, a timely and thorough coverage of the versatility and opportunities provided by peptide and phage probes in electrochemical biosensing based on selected reports mainly from the last 2 years is accomplished.

Peptide probes

Peptides, short chain-like polymers containing less than 50 amino acids in length connected by peptide bonds, are star probes which have experienced an unstoppable boom in the development of electrochemical biosensing strategies with improved performance [7, 13,14,15,16, 20, 21]. Their use as probes in electrochemical biosensing is advantageous due to their small size, high affinity, stability, structural and sequence diversity, biocompatibility, facile processability, and lower immunogenicity compared with antibodies. They can be easily obtained with high yield and affordable cost as well as modified with specific functional groups for immobilization or signaling through automated chemical synthesis, avoiding the need for laborious in vivo procedures and animal immunization to reduce the use of laboratory animals and follow the EU recommendations on animal protection and replacement of animal-derived antibodies by non-animal-derived ones [22], and displaying higher chemical stability than antibodies [13, 23,24,25]. On the other hand, compared with nucleic acid aptamers, peptides have smaller binding regions and variable surface charges and are feasible for protease-based assays as natural substrates. Moreover, peptides provide a varied cross-linking methodology with the biosensing interface. For example, they can be immobilized on a gold surface through Au–S bonding using the cysteine thiol group, or they can be covalently immobilized by binding to carboxyl/amino group-functionalized interfaces through carbodiimide/succinimide chemistry [15, 16, 26].

Due to their versatility of modification and use, flexible variability, tuneable properties, and multifunctionality, peptides and their derivatives (complexes [27, 28], hydrogels [29, 30], nanotubes [15], nanoparticles [26], etc.) have been used in electrochemical biosensing as [7, 13,14,15,16, 31]:

Interfacial materials (electrode modifiers) or self-assembled units/nanostructures (to immobilize other receptors in a suitable arrangement) to impart particular properties (antibiofouling, biocompatibility) and/or improve the biosensing performances;

Recognition ligands to interrogate a wide variety of analytes;

Enzymatic substrates (e.g., proteases and kinases);

Enzyme mimics; and

Signaling elements/carriers.

Due to their distinguished properties, antibiofouling, multifunctional, multimeric, and switching, peptides have gained special importance in recent years in electrochemical biosensing. Table 1 summarizes representative examples of methods developed during the last 2 years.

As can be deduced from Table 1, peptides have been exploited primarily as recognition elements [32, 33, 35, 37, 41], electrode modifiers [24, 26, 30, 39, 40], modifiers of other probes [34, 43], enzymatic substrates [36], mimicked enzymes [27, 28], and tracers [38, 42] for the electrochemical biosensing of a wide variety of targets including foodborne pathogens [32], immunoglobulins [33, 39], viral antigens [24, 38], cells [35], tumor markers [26, 27, 30, 34, 36, 37, 42, 43], pesticides [28], and antibiotics [40, 41].

In general, although the affinity of avidin/streptavidin for biotin has also been used for their attachment [34, 36], peptides have been immobilized on gold or nanostructured with AuNPs electrode surfaces through their self-assembly profiting the gold-thiol chemistry [24, 30, 33, 35, 37, 39, 40, 42]. In the photoelectrochemical (PEC) platform reported by Yin et al. [32], an antimicrobial peptide was immobilized on a flexible paper substrate modified with core-shell-structured upconversion nanophosphors ((UCNPs)@SiO2@Ag) and carbon self-doped graphitic carbon nitride (C-g-C3N4). A different approach has recently been described by Chen et al. [43], who fabricated a ternary photoelectrode by modifying a hydrogen-bonded organic framework (HOF-101) and polydopamine (PDA) onto a ZnO array electrode where a branched zwitterionic peptide (BZP) linked to complementary DNA (cDNA) through a click reaction was anchored.

Particularly relevant is the use of peptides as electrode modifiers [24, 26, 30, 39, 40], conjugated with other recognizing probes [34, 43], or as multifunctional bioreceptors [33, 35, 41] to implement fouling-free electrochemical biosensing strategies.

Peptides have been used as electrode modifiers in the development of affinity biosensors [24], immunosensors [30, 39], and aptasensors [26, 40, 43] with antifouling properties which have been applied to the determination of SARS-CoV-2 receptor-binding domain (RBD), IgM, PSA, TC, mucin-1 (MUC1), and carcinoembryonic antigen (CEA) in milk, blood, and serum samples. Among these peptides, zwitterionic peptides [26, 30, 40, 43], those involving D-amino acids [39], and cyclic peptides [24] stand out, the last two types showing an outstanding proteolytic resistance, thus overcoming one of the main complications faced by the proper functioning of peptide biosensors in complex environments [39]. For example, Han et al. [24] recently proposed a biosensor for the determination of the RBD of the SARS-CoV-2 spike glycoprotein by modifying a GCE/PEDOT/AuNPs with a cyclic peptide to impart self-fouling properties to the surface, and angiotensin-converting enzyme 2 (ACE2) as a target recognition element (Fig. 2). Due to the stable structure of the designed cyclic peptide and the absence of any N- or C-terminal amino acids, this biosensor exhibited noticeable resistance to biofouling and enzymatic hydrolysis even in human blood, thus enabling the accurate determination of the target in this complex matrix.

Fig. 2

Biosensor for the determination of RBD of SARS-CoV-2 spike glycoprotein exploiting the use of a cyclic peptide as an electrode modifier. Reproduced from [24] with permission from the Royal Society of Chemistry

It is worth drawing attention to the low fouling and highly sensitive electrochemical biosensor reported by Chen et al. [34] for the determination of CA125 involving antifouling peptide-DNA conjugates formed through a reagent-free click reaction (Fig. 3). The biosensor was able to analyze CA125 in undiluted human serum and provided a universal strategy to prepare antifouling biosensors through the conjugation of the antifouling peptides with the specific DNA probes. In addition, a new PEC aptasensor was developed recently and applied to the analysis of MUC1 in human serum [43].

Fig. 3

Electrochemical biosensor developed for the determination of CA125 involving the use of antifouling peptide-DNA conjugates. Reprinted from [