www.advmattechnol.de 2200310 (1 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH PersPective Empowering Electrochemical Biosensing through Nanostructured or Multifunctional Nucleic Acid or Peptide Biomaterials Susana Campuzano,* María Pedrero, Rodrigo Barderas, and José M. Pingarrón* DOI: 10.1002/admt.202200310 at the forefront of modern detection tech- niques. This is due to their high selec- tivity and sensitivity, ease of use and low cost, fast response time, and feasibility to operate at the multiplexed and multi- omics level either in centralized or field settings owing to the possibility to work both with complex instruments and with simple and portable devices.[1] The great advances demonstrated by electrochem- ical biosensors in recent years have gone hand by hand with the development of new electrochemical substrates, attractive surface chemistries, bioassay formats, and amplification strategies, but also with the production and application of new nano- materials and bioreceptors, which has made possible to generate superb devices capable of facing challenging applications. In particular, the design and exploita- tion of nanostructured or multifunctional nucleic acid or peptide biomaterials has proven to empower the electrochemical biosensors performance in terms of sensitivity, storage stability, response time, simplicity, and robustness, making the move of these devices from the research laboratory to the marketplace does not seem like science fiction. As discussed in more detail in the following sections, the use of state-of- the-art nanostructured or multifunctional nucleic acid or peptide biomaterials has contributed compelling answers to important challenges conditioning the performance of electrochemical biosensors, among which it is worth intro- ducing at this point to put the reader in context: i) Controlling the density and orientation of recognition nu- cleic acid or peptide probes arranged on heterogeneous sur- faces, especially in comparison with the affinity reactions in homogeneous solution, particularly those employing single- stranded DNA capture probes due to their lack of stiffness, which critically conditions the sensitivity, reproducibility, and stability of the resulting biodevices.[2–4] This goal can be at- tained by exploiting in a rational way DNA-, aptamer-, and peptide-based nanofabrication, enabling the preparation of highly controlled and precise programmable geometries.[4] ii) Ensuring the reliability and robustness of the analytical results and their real-world applicability by making electrochemical biosensors less susceptible to the electrode surface state, probe packing density, environment and artificial factors, Electrochemical biosensors continue to evolve at an astonishing pace, con- solidating as competitive tools for determining a wide range of targets and relentlessly strengthening their attributes in terms of sensitivity, selectivity, simplicity, response time, and antifouling ability, making them suitable for getting a foothold in real-world applications. The design and exploitation of nanostructured or multifunctional nucleic acid or peptide biomaterials are playing a determinant role in these achievements. With the aim of high- lighting the potential and opportunities of these biomaterials, this perspec- tive article critically discusses and overviews the electrochemical biosensors reported since 2019 involving nanostructured and multifunctional DNA biomaterials, multifunctional aptamers, modern peptides, and CRISPR/Cas systems. The use of these biomaterials as recognition elements, electrode modifiers (acting as linkers or creating scaffolds with antifouling properties), enzyme substrates, and labeling/carrier agents for signal amplification is discussed through rationally and strategically selected examples, concluding with a personal perspective about the challenges to be faced and future lines of action. S. Campuzano, M. Pedrero, J. M. Pingarrón Department of Analytical Chemistry Faculty of Chemistry Complutense University of Madrid Madrid 28040, Spain E-mail: susanacr@quim.ucm.es; pingarro@quim.ucm.es R. Barderas Chronic Disease Programme UFIEC Institute of Health Carlos III Majadahonda, Madrid 28220, Spain The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/admt.202200310. 1. Introduction to Electrochemical Biosensors and Their Current Empowerment It is indisputable that electrochemical biosensors continue to establish themselves as very promising analytical tools and are © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Adv. Mater. Technol. 2022, 2200310 http://crossmark.crossref.org/dialog/?doi=10.1002%2Fadmt.202200310&domain=pdf&date_stamp=2022-04-28 www.advancedsciencenews.com www.advmattechnol.de 2200310 (2 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH and the nature of the samples. All this can be approached by developing dual-signal ratiometric strategies and antifouling devices.[5,6] iii) Improving sensitivity and reducing assay time by resorting to the clever use of nanostructured or multifunctional bioma- terials of nucleic acids or peptides as recognition elements, clinically relevant enzyme substrates, electrode modifiers (such as linkers and scaffolds), and labeling/transport agents. Also noteworthy is the coupling of such biomaterials with other types of nanomaterials and their use as “dispersible electrodes” capable of bringing the sensor to the analyte instead of the ana- lyte encountering the sensor (conventional paradigm).[7] Enlightened by the above, the purpose of this perspective article is to offer the reader an overview of the latest advances and opportunities provided by nanostructured or multifunc- tional biomaterials based on nucleic acids or peptides in electrochemical biosensors, with the purpose of giving the sci- entific community reasons to explore them in a simple and/or combined way with artificial nanomaterials. To our knowledge, this timely and appropriate topic for the research community has not yet been covered in the literature. This is done by com- prehensively presenting a selection of representative recent works (mostly since 2019), being aware of the impossibility to fully cover the immense number of reported approaches. More- over, critical, and somewhat objective views on the challenges to be met to move the field forward and the future landscape of these biodevices is given. 2. Nanostructured and Multifunctional DNA Biomaterials DNA is an excellent self-assembly nanomaterial due to its fore- seeable base pairing, high chemical stability, and convenience of synthesis and modification. These unique properties explain its widespread use in electrochemical biosensors, both as a rec- ognition element or multifunctional biomaterial (polyA and DNAzyme probes) and/or to generate unique nanoassemblies, including nanostructures (Y-shaped, tetrahedral, origami, and dendrimers, nanostructures and nanospheres), networks, and hydrogels.[2,8] Table 1 summarizes the main characteristics of selected representative electrochemical (bio)sensors developed mostly since 2019 involving nanostructured and multifunctional DNA biomaterials. 2.1. DNA Nanostructures DNA nanostructures have been employed as recognition ele- ments, electrode modifiers, and for amplification purposes. It is important to note that, although they generate DNA nanostruc- tures to some extent, triplex DNA nanostructures and electro- chemical biosensors using proximity ligation assays (PLA)[38,39] are considered outside the main scope of this perspective article. The PLA strategy is based on DNA ligation of two adja- cent probe oligonucleotides by bivalent binding. PLA has been shown to be functional with both aptamer and antibody pairs and has demonstrated potential in ÷ ÷ for detection of both genetic and protein targets.[40] This strategy has undergone sev- eral modifications to overcome its limitations, among which, the proximity extension assay should be highlighted. It being based on the extension of hybridizing oligonucleotides with DNA polymerase, has been shown to improve efficiency com- pared to PLA in biological samples such as plasma.[41] 2.1.1. Y-Shape, Tetrahedral, Origami, and Dendrimers Nanostructures Y-shape DNA nanostructures, composed of three oligonucleo- tides, which partially hybridize to each other, have been shown to impart interesting properties to electrochemical biosen- sors.[17] The use of a “Y” shaped structure allows the probe to be stably fixed at the electrode surface and keeps it upright at the interface, this allowing a higher recognition efficiency com- pared to straight ones, particularly for long targets, also mini- mizing non-specific adsorptions.[5,42] Y-shape DNA nanostructures have been exploited in devel- oping biosensors for the determination of relevant nucleic acids (DNAs, miRNAs, lncRNAs, and mRNAs) achieving LODs at the fM level for short DNAs[19] and RNAs[5,17,16] and pM for the determination of long RNAs.[18] These biosensors were applied to the analysis of serum or total RNA samples extracted from cells, exosomes, and urine. Importantly, the high sensitivity exhibited was reached by combining these Y-shape nanostruc- tures with amplification strategies such as mismatched cata- lytic hairpin assembly (CHA),[16] non-linear hybridization chain reaction (HCR),[17] assisted strand displacement reaction,[5] and CHA and terminal deoxynucleotidyl transferase (TdT) catalysis.[19] It is also noteworthy that two of these biosensors employed ratiometric readout.[5,16] Figure 1 shows the electrochemical biosensor schemes based on the use of Y-shape DNA nanostructures as electrode modi- fiers for the determination of a short RNA (Figure 1a) and two types of longer RNA: lncRNA and mRNA (Figure 1b).[16,18] The use of tetrahedral and origami DNA nanostructures in electrochemical biosensors has been proposed as efficient alternatives to improve the recognition capability of surface- confined heterogeneous DNA probes due to control of surface chemistry, conformation, and packing density of probe mole- cules, and surface size and geometry.[2] The introduction of tetrahedral DNA nanostructures (TDNs, also known as tetrahedral tripods (TTs) or tetrahedral-structured probes (TSPs)) in electrochemical biosensors allows controlling the spacing and density of the immobilized probe, improving the stability of the biosensor (three anchor points instead of the single point of the single-stranded DNA capture probes) and the target accessibility, as well as imparting the biodevice with anti- fouling properties.[2,3,9,10] The TDNs scaffolds are easily, rapid, and reproducibly formed by a single-step self-assembly of four probes (three of them thiolated) onto gold surfaces, without requiring the use of mercaptohexanol (MCH) to lift the capture probe and prevent nonspecific adsorptions.[2,43,44] They exhibit an excellent mechanical rigidity and structural stability, imparting stability to the detection process, and have demonstrated to greatly improve the sensitivity, selectivity, and storage stability of the resultant electrochemical biosensors.[2–4] It is important to note that the TSP-modified surfaces are fully compatible with Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (3 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH Table 1. Representative recent electrochemical (bio)sensors exploiting the use of nanostructured and multifunctional DNA biomaterials. Electrode Fundamentals Biomaterial Detection technique Target analytes LR/LOD Sample/application Remarkable features Ref. DNA nanostructures: Tetrahedral, origami, dendrimers, Y-shape nanostructures, and nanospheres AuE TSPs comprising a sequence complementary to the target miRNA and a G-quadruplex sequence. In the presence of the target miRNA the DSN hydrolyzed the DNA in the formed DNA-RNA heteroduplexes TSPs + DNAzyme DPV (Hemin/ H2O2/L-cysteine) Signal-off miRNA-21 0.1 fm–0.1 pm/ 0.04 fm Spiked human serum Ultrasensitive due to triple signal Amplification: TSPs + DSN + G-quadruplex−hemin conformation [3] AuE TSPs in combination with proximity dependent surface hybridization and redox reporter modified probes TSPs + PLA DPV (Fc) Signal-on DNA 1 fm–10 pm/0.2 fm Spiked bovine serum and Listeria monocytogenes extraction solution Label-free [4] GCE modified with AuNPs and NP-rGO TSPs in combination with RCA TSPs DPV (HRP/H2O2/ HQ) Signal-on Thrombin 1 × 10−13–1 × 10−7 m/ 3.53 × 10−14 m Spiked human serum – [9] AuNPs-SPEs Sandwich hybridization assay in connection with AuNPs decorated with reporter probes and HRP for amplification purposes TSPs Amperometry (TMB) BRCA1 1 fm–1 nm/0.1 fm – – [10] AuE Use of TTs and MTHsA TSPs DPV (RuHex) Signal-on DNA, microRNA and methylated DNA DNA: 1 am– 100 pm/0.59 am Spiked human serum Ultrasensitive [11] ITO Dual aptamer-modified Fe3O4 −@SiO2 and hairpins functionalized TSPs in com- bination with hyperbranched HCR TSPs DPV ([Ru(NH3)6]3+/ [Fe(CN)6]3−) Ru3+: Signal-off Fe3+: Signal-on Exosomes 5 × 103 particles mL−1 Serum samples from breast cancer patients Ratiometric [12] AuE Direct hybridization of the target miRNA at DNA origami probes immobilized on the WE via electrostatic adsorption onto a chitosan film DNA origami nanostructures DPV (MB) Signal-on miRNA-21 0.1 pm–10.0 nm/ 79.8 fm Spiked human serum Label-free, amplification-free [13] AuE Displacement assay based on direct hybridization of the target DNA at redox reporter modified, surface bound triangle origami receptors DNA origami nanostructures DPV (MB) Signal-off Target DNA – – Label-free, amplification-free [14] GCE PLCHA, PtNPs-function- alized single strands and functional DNA dendrimers which remarkably facilitates the entrapping of MnTMPyP. DNA dendrimers SWV (H2O2/4-CN) Signal-on Thrombin 1 fm–100 nm/ 10.7 am Spiked human serum – [8] GCE Sandwich immunoassay using a cDNA-labeled secondary antibody, a couple of complementary Y-shaped DNAs, and a DNA dendrimer for high loading of MB molecules DNA dendrimers and Y-shape nanostructures SWV (MB) Signal-on PSA 1 pg mL−1–10 ng mL−1/0.26 pg mL−1 Serum samples – [15] AuE Mismatched CHA amplifica- tion, Fc- and MB-tagged hairpin probes, and forma- tion of “Y”-shaped DNA complexes Y-shape nanostructures DPV (MB, Fc) Fc: Signal-on; MB: Signal-off miRNA-21 5 fm–0.1 nm/1.1 fm RNAt extracted from cells Ratiometric readout, self-calibrating [16] Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (4 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH Electrode Fundamentals Biomaterial Detection technique Target analytes LR/LOD Sample/application Remarkable features Ref. AuE Y-shape probes and HCR Y-shape nanostructures DPV (Fe(CN)6]−3/−4) Signal-off miRNA-25 1 fm–10 pm/0.3334 fm Spiked human serum Label-free Discrimination of single base mutations [17] PLLy/GCE Direct hybridization, Fc- and MB-tagged probes and assisted strand displace- ment reaction with a LNA-modified “Y” shape-like structure Y-shape nanostructures DPV (MB, Fc) Fc: Signal-off; MB: Signal-on Exosomal miRNA-21 10–70 fm/2.3 fm RNAt extracted from exosomes Ratiometric readout, regenerative [5] Au-SPEs Sandwich hybridization assay using Y-shaped capture probes and multiple 6-FAM-tagged reported probes Y-shape nanostructures Amperometry (HRP/TMB/H2O2) Signal-on PCA3 lncRNA and PSA mRNA PCA3 lncRNA: 25 pm–10 nm/4.4 pm PSA mRNA: 25 pm–1 nm/1.5 pm RNAt extracted from cells and urine Dual determination [18] AuE Direct hybridization, CHA, and TdT catalysis Y-shape nanostructures DPV (MB) Signal-on HPV16 oncogene 2 fm–10 pm/0.19 fm Spiked human serum – [19] TiO2-modified GCE The target-related ternary “Y” structure cleavage cycling reaction trigger RCA to in situ form PDA+- decorated multifunctional DNA spheres on the TiO2 substrate DNA nanospheres DPV (PDA+) Signal-on miRNA-141 PEC: 0.1 fm–1 nm EC: 2 fm–500 pm – Dual PEC and EC detection mode [20] DNA networks, hydrogels, and PLA assays AuE MB-DNA-Au@MNPs DNA network “dispersible electrode” SWV (MB) Signal-on miRNA-21 10 am–1 nm RNAt extracted from cancer cells, raw serum, and 50% blood 30 min assay time. Ultrasensitive. 8 orders of magni- tude linear range. Detection in unprocessed blood samples. [21] AuE MB-DNA-Au@MNPs DNA network “dispersible electrode” SWV (MB) Signal-on ctDNA 22 nts DNA target: 2 am–20 nm/3.3 am 101 nts ctDNA target indicative of NSCLC: 200 am–20 nm/5 fm Spiked undiluted human plasma and 50% whole human blood Detect short- and long strand DNA targets. 20 min response time. Robustness in complex biological media. [22] AuE Elongation of a specific primer and use SA-biotin- DNA-biotin networks for amplification DNA network EIS (Fe(CN)6]−3/−4) Signal-on Telomerase 50–5000 cell mL−1/2 cells Cells PCR-free and enzyme-free. Feasibility to perform telomerase inhibition studies. [23] AuE Proximity binding-induced DNA network assembled by non-linear HCR DNA network DPV (MB) Signal-on Thrombin 1.0 pm–1.0 nm/0.56 pm Spiked human serum Enzyme-free and ultrasensitive [24] AuNPs/GCE Target-induced DNA hydrogel formation with pH-stimulated signal amplification DNA hydrogel EIS (Fe(CN)6]−3/−4) Signal-on HPA 0.01 pg mL−1–20 ng mL−1/0.003 pg mL−1 Spiked human serum – [25] AuNPs/GCE DNA hydrogel by coupling with DNAzyme-assisted target recycling and HCR DNA hydrogel EIS (Fe(CN)6]−3/−4) Signal-on Hg2+ 0.1 pm–10 nm/0.042 pm Spiked tap water samples – [26] ITO DNA hydrogel to enwrap enzymes (HRP, BOD) DNA hydrogel (pure) CV (H2O2/TH) H2O2 30 nm–100 µm/10 nm – Regeneration and reusability. Feasible colorimetric and electrochemical detection [27] Table 1. Continued. Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (5 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH Electrode Fundamentals Biomaterial Detection technique Target analytes LR/LOD Sample/application Remarkable features Ref. ITO/PET DNA hydrogel and Fc- tagged probes DNA hydrogel (Hybrid) DPV (Fc) Signal-off miRNA-21 10 nm–50 µm/5 nm – Good selectivity and storage stability [28] ITO DNA hydrogel with TBO and G-quadruplex/hemin DNAzyme DNA hydrogel (Pure) CV (H2O2) Signal-on H2O2 0.2–80 µm/0.15 µm – Anti-interference ability and recon- struction ability [29] Multifunctional DNA probes 16-channel AuE chip Sandwich hybridization assays using a polyA-capture probe and two biotin-labeled reporter probes PolyA-probes Chronoamperom- etry (HRP/TMB/ H2O2) Signal-on Five bacterial 16S rRNA (St, En, Es, Ps, and Ci) St: 10 fm–1 nm/5 fm Staphylococcus aureus genomic DNA Reusability. Multiplexed system. [30] AuE Direct hybridization at a triblock DNA capture probe (PAP) PolyA-probes Chronoamperom- etry (HRP/TMB/ H2O2) Signal-on DNA 10 fm–1 nm/10 fm Asymmetric PCR amplicon from E. coli genomic DNA Label-free. Discrimination of single nucleotide polymorphisms [31] AuE Sandwich hybridization using a polyA-capture probe, 12 reporter probes, and 2 spacers PolyA-probes Chronoamperom- etry (HRP/TMB/ H2O2) Signal-on Transgene- derived long RNA (RNAi GMO) Long stem-loop RNA: 1 pm–300 nm/1 pm (10 am, for a 379 bp dsDNA RT-RPA amplicon) RNA from RNAi- based transgenic maize leaves Detect long RNAs [32] AuE Strategy combining DNA walker (three legs DNAzyme spiders) with PLA DNAzymes SWV (Fc) Signal-on Telomerase 25–2000 cells/10 Hela cells Enzyme-free, PCR-free [33] GCE Bifunctional DNAzyme nanodevice with two detec- tion paths toward the same target DNAzymes DPV (MB, Fc) MB: Signal-on; Fc: Signal-off Hg2+ 0.1 pm–200 nm/23 fm Spiked tap water Ratiometric [34] CNTs-SPE Isothermal signal amplifica- tion via the polymerization/ nicking reaction and DNA- zyme-catalyzed cleavage of Fc-DNA from PES (prepared by assembling Fc-DNA functionalized paper and CNTs-SPE) DNAzymes DPV (Fc) Signal-on miRNA-21, ALP, and CEA miRNA-21: 1 fm–1 µm ALP: 1–105 mU L−1 CEA: 1–1500 fg mL−1 miRNA-21: Spiked human serum PES zero-background current. Universal potential for POC diagnosis in resource-limited settings [35] Nanostruc- tured AuE Electroactive RNA-cleaving DNAzymes (e-RCDs) inte- grated into a two-channel electrical chip with nano- structured electrodes DNAzymes DPV (MB) Capture channel: Signal-on; release channel: Signal-off E. coli 10 CFU Urine samples Differential electro- chemical signaling. Analysis time < 1 h. Reagent-free [36] SPE Zr-MOFs-modified paper and aptamer as the recogni- tion system and HCR and DNAzyme-mediated catal- ysis for signal amplification DNAzymes DPV (TMB) Signal-on Exosomes 5 × 103 particles mL−1 Spiked FBS Label-free Enzyme-free Paper-based biosensor [37] ALP: Alkaline phosphatase; AuE: gold electrode; AuNPs: gold nanoparticles; BOD: bilirubin oxidase; CEA: carcinoembryonic antigen; CFU: colony forming units; CHA: catalytic hairpin assembly; Ci: Citrobacter f reundii; 4-CN: 4-chloro-1-naphthol; CNTs: carbon nanotubes; ctDNA: circulating tumor DNA; CV: cyclic voltammetry; DNA-Au@ MNPs: network of probe DNA modified gold-coated magnetic nanoparticles; DPV: differential pulse voltammetry; DSN: duplex-specific nuclease; EC: electrochemical; EIS: electrochemical impedance spectroscopy; En: Enterococcus faecalis; e-RCDs: electroactive RNA-cleaving DNAzymes; Es: Escherichia coli; 6-FAM: 6-carboxyfluorescein; FBS: fetal bovine serum; Fc: ferrocene; GCE: glassy carbon electrode; GMO: genetically modified organism; HCR: hybridization chain reaction; HPA: heparanase; HPV16: human papillomavirus 16; HQ: hydroquinone; HRP: horseradish peroxidase; ITO: indium tin oxide; ITO/PET: indium tin oxide/polyethylene terephthalate; LNA: locked nucleic acid; LOD: limit of detection; LR: linear range; MB: methylene blue; MnTMPyP: manganese(III) meso-tetrakis (N-methyl-pyridinium-2-yl)-porphyrin; MTHsA: multiple tandem hairpins assembly; NP-rGO: N- and P-co-doped Graphene; NSCLC: non-small-cell lung cancer; nts: nucleotides; PAP: probe−PolyA−probe; PCA3: prostate cancer antigen 3; PCR: polymerase chain reaction; PDA+: N,N-bis(2-(trimethylammonium iodide)propylene)-perylene-3,4,9,10-tetracarboxydiimide; PEC: photoelectrochemical; PES: paper-based electrochemical sensor; PLA: proximity ligation assay; PLCHA: proximity ligation-responsive catalytic hairpin assembly; PLLy: polylysine membrane; POC: point of care; Ps: Pseudomonas aeruginosa; PSA: prostate specific antigen; PtNPs: Pt nanoparticles; RCA: rolling circle amplification; RNAi: RNA interference; RNAt: total RNA; RT-RPA: reverse transcription recombinase polymerase amplification; RuHex: hexaammineruthenium(III) chloride; SA: streptavidin; SPE: screen-printed electrode; St: Staphylococcus aureus; SWV: square wave voltammetry; TBO: toluidine blue O; TdT: terminal deoxynucleotidyl transferase; TH: thionine; TMB: 3,3′,5,5′-tetramethylbenzi- dine; TSPs: DNA tetrahedron-structured probes; TTs: tetrahedral tripods; WE: working electrode. Table 1. Continued. Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (6 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH electrochemical interrogation. Despite the relative thickness of the TSP layer (≈6 nm), its hollow structure allows easy diffusion of the electroactive species to the modified electrode surface.[2] It is important to note that although they have been much less used than as electrode modifiers, TSPs have also been exploited for amplification purposes by taking advantage of the six edges they have to bind numerous electroactive molecules.[12] Table 1 includes the characteristics of some recently reported TDNs-based electrochemical biosensors for determining a broad range of molecules (proteins, DNAs, miRNAs, methyl- ated DNAs, and tumor exosomes). Importantly, these strate- gies combine the use of these DNA nanostructures with that of nanomaterials, DNAzymes, and other amplification strate- gies (PLA, duplex-specific nuclease (DSN), rolling circle ampli- fication (RCA), multiple tandem hairpins assembly (MTHsA), and HCR) to achieve sensitivities at the am–fm level of nucleic acids, fm of proteins, and 104 exosomes mL−1. A representative example is the electrochemical biosensor reported by Lu et al. for the determination of miRNA-21.[3] As can be seen in Figure 2a), the authors designed and used as electrode modifiers TDNs composed of a sequence able to recognize the target miRNA and a G-quadruplex sequence that, combined with hemin, mimicked peroxidase activity for H2O2 reduction and L-cysteine oxidation. A DSN hydrolyzed the DNA–RNA heterohybrids generated in the presence of the target miRNA releasing this (which can act in a next cycle) and the G-quadruplex sequence, which resulted in a decrease in the differential pulse voltammetric (DPV) response pro- portional to the concentration of the target miRNA. The biosensor achieved a LOD of 0.04 fm, good selectivity even for a single-base-mismatched sequence and was applied to determine the target miRNA in serum samples from breast- cancer patients providing results in line with those obtained by qRT-PCR. Figure 1. Y-shape DNA nanostructure-based electrochemical biosensors for the determination of a) miRNA-21 and b) PCA3 (lncRNA) and PSA mRNA. (a) Reproduced with permission.[16] Copyright 2019, Elsevier; (b) Reproduced with permission.[18] Copyright 2021, Elsevier. Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (7 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH On the other hand, Figure 2b illustrates the ratiometric strategy proposed by Yang et al. for the highly sensitive determination of tumor exosomes by using dual aptamer-modified Fe3O4 −@SiO2 and hairpins functionalized TSPs in combination with hyper- branched HCR for amplification purposes.[12] In connection with CD63 and mucin 1 (MUC1)-specific aptamers this strategy allowed the detection of as low as 3 × 104 particles mL−1 and differ- entiation of breast cancer patients from healthy individuals. In recent years DNA origami nanostructures have aroused great interest due to their diverse structural engineering capa- bilities, large surface area, and unprecedented customization to precisely organizing the target binding sites at the nanoscale. Similarly to TDNs, this type of nanostructures allows the immobilization of multiple single-stranded DNA probes at desired locations thus increasing their accessibility and recog- nition efficiency due to the rational controlled density of DNA Figure 2. Electrochemical biosensors for the determination of a) miRNA-21 and b) exosomes based on the use of TDNs coupled with DSN (a) and HCR (b) as electrode modifiers and for amplification purposes, respectively. (a) Adapted with permission.[3] Copyright 2019, Elsevier; (b) Reproduced with permission.[12] Copyright 2021, ACS. Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (8 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH probes. However, DNA origami nanostructures avoid the use of thiolated probes and, because of their larger surface area, allow the immobilization of a larger number of probes compared to TDNs.[13,14] Nevertheless, to date the use of DNA origami nano- structures in electrochemical biosensors is much smaller than that of TDNs. An illustrative example of using DNA origami as electrode modifier is the method proposed by Han et al. reporting a label- and amplification-free electrochemical biosensor for miRNA-21 by physically adsorbing cross-shaped DNA origami nanostruc- tures containing protruding ssDNA probes on a chitosan film and using methylene blue (MB) as a redox indicator of the hybridization process (Figure 3).[13] The biosensor attained a LOD as low as 79.8 fm, was able to discriminate single-base mismatched sequences, and exhibited satisfactory performance in spiked human serum samples. DNA dendrimers (DNADS) are highly branched nanostruc- tures formed by sequential complementary hybridization of pre-designed DNA components that can be easily employed to stably anchor a large number of signal amplifiers (small mole- cules, biomolecules, or metal nanoparticles) allowing amplifica- tion of the electrochemical response, improving the sensitivity and extending the linearity range of the resulting electrochem- ical biosensors.[8,15] For instance, Liao et al. reported an aptasensor for the sensi- tive determination of thrombin where they resorted to proximity ligation CHA (PLCHA) to guide the construction of DNADS trapping Pt nanoparticles (PtNPs) and manganese(III) meso- tetrakis(N-methyl-pyridinium-2-yl)-porphyrin (MnTMPyP) with pseudoperoxidase activity, they acting as synergistic enhancers of the 4-chloro-1-naphthol (4-CN) oxidation in the presence of H2O2 (Figure 4a).[8] Another DNADS self-assembled by hybridization of a pair of complementary oligonucleotides, covalently conjugated to three arms of a Y-shaped crosslinker, tris(2-maleimidoethyl)amine, was employed to amplify the electrochemical signal of an immunosensor for the determination of PSA.[15] As Figure  4b shows, this immunosensor involved a sandwich immunoassay format and the use of a complementary oligonucleotide (cDNA)- labeled detection antibody to bind the DNA dendrimer acting as carrier to load multiple electroactive MB molecules leading to the amplification of the electrochemical response. It is impor- tant to highlight the amazing sensitivity of these electrochem- ical biosensors with LOD values of 10.7 am and 0.26 pg mL−1 for the determination of thrombin and PSA, respectively.[8,15] 2.1.2. Nanospheres An illustrative work of these DNA nanostructures is the dual photoelectrochemical and electrochemical detection carried out with the biosensor developed by Deng et  al. for miRNA-141 determination.[20] The method involved the in situ generation of cationic N,N-bis(2-(trimethylammonium iodide) Figure 3. Electrochemical biosensor for the determination of miRNA-21 based on the use of a gold electrode modified with cross-shaped DNA origami nanostructures. Reproduced with permission.[13] Copyright 2019, ACS. Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (9 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH Figure 4. Electrochemical aptasensor and immunosensor for the determination of a) thrombin and b) PSA exploiting the use of DNADS to attach a large number of PtNPs + MnTMPyP (a) or MB molecules (b), respectively, for signal amplification purposes. (a) Reproduced with permission.[8] Copyright 2020, Elsevier; (b) Reproduced with permission.[15] Copyright 2021, Elsevier. Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (10 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH propylene)-perylene-3,4,9,10-tetracarboxydiimide (PDA+)-deco- rated multifunctional DNA nanospheres on the electrode sur- face. In this bioplatform a target-related ternary “Y” structure cleavage cycling reaction converted the target DNA into mas- sive output DNA anchored on a TiO2 substrate, which triggers an RCA reaction. The long DNA tails of the RCA product con- densed in situ in the presence of Mg2+ and PDA+, forming the multifunctional DNA spheres (Figure 5). This biosensor dis- played linearity between 2 fm and 500 pm. 2.2. DNA Networks and Hydrogels The use of DNA networks and hydrogels has shown to be par- ticularly interesting to empower the sensitivity of electrochem- ical biosensors. DNA networks have been exploited to improve sensitivity and reduce assay time through the concept of using conductive gold- coated magnetic nanoparticles as “dispersible electrodes.” as pro- posed by Gooding´s Group,[7,21,22,45] and for amplification using more conventional electrochemical biosensor formats.[23,24] The key to “dispersible electrodes” is the use of Au-coated MNPs (Au@MNPs), modified with the appropriate receptor to selectively recognize the analyte, combining the ability to move the NPs with a magnetic field with electrical conductivity. This strategy showed a 1000-fold improvement in both detection limit and response time compared to the same sensing ele- ments in macroscopic and planar electrochemical sensors.[7] Taking advantage of this concept, electrically reconfigurable networks of DNA-Au@MNPs have been employed for ultra- sensitive (in the am range) and fast (20–30 min) detection of miRNAs and circulating tumor DNA (ctDNA).[21,22] Figure 6 illustrates the strategy reported by Gooding’s team for the determination of ctDNA that combines the use of Au@MNPs modified with a DNA probe dually labeled with thiol and MB (MB-DNA-Au@MNPs) and complementary to the target ctDNA, with the use of a gold macroelectrode modified with a binary monolayer of a single-strand DNA (also dually end-labeled with thiol and MB) and MCH to achieve signal amplification through the electroreduction of ferricyanide by MB tags from both surfaces (electrode and Au@MNPs). As Figure 6a shows, the MB-DNA-Au@MNPs were exposed to the target ctDNA and the hybridized MB-DNA-Au@MNPs were collected with the aid of a magnet onto the gold electrode sur- face to monitor electrochemically a redox amplification cycle in which MB is reduced to leucomethylene blue (LB) which Figure 5. Electrochemical and photoelectrochemical-dual mode biosensor for the determination of miRNA-141 by utilizing multifunctional DNA nanospheres and cleavage cycling amplification and RCA strategies. Reproduced with permission.[20] Copyright 2020, Elsevier. Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (11 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH then oxidizes back to MB (oxidation recorded by square wave voltammetry (SWV)) by [Fe(CN)6]3−. The decrease in the SWV signal observed in the presence of target ctDNA was attributed to the greater distance between the MB-DNA-Au@MNPs after hybridization which hindered the electron tunneling through the MB-DNA-Au@MNPs network.[21] This strategy allowed the determination of target DNAs of different length (22–101 nts) in raw human plasma and 50% whole human blood. DNA networks generated by DNA probes functionalized with biotin at both ends[23] or by non-linear HCR amplification strategies[24,46] (Figure  6b) have been exploited to develop bio- sensors with improved sensitivity. Representative examples of Figure 6. DNA networks used a) in “dispersible electrodes” for the ultrasensitive and rapid detection of ctDNA and b) generated by non-linear HCR for amplification purposes in the determination of thrombin. (a) Reproduced with permission.[22] Copyright 2021, RSC; (b) Reproduced with permis- sion.[24] Copyright 2021, Elsevier. Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (12 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH these concepts are the enzyme-free biosensors reported by Liu et al. for the determination of telomerase activity and by Li et al. for thrombin.[23,24] DNA hydrogels are 3D porous network polymers containing a large amount of water and constructed by crosslinking only nucleic acids (pure DNA hydrogels) or grafting DNA strands on hydrophilic polymers or other materials (hybrid DNA hydro- gels).[26] Pure DNA hydrogels have limited mechanical proper- ties and high cost compared to the hybrid ones.[28] Although they have not yet been largely explored, DNA hydrogels are considered as promising biomaterials for elec- trochemical biosensors because of their biocompatibility, flex- ibility, large specific surface area and loading capacity, high diffusivity of small molecules, mechanical stability, and ability to respond to appropriate stimuli (such as pH, light, etc.).[25] These biomaterials have been employed both as electrode modifiers, providing 3D scaffolds to enwrap catalytic or affinity receptors or 3D electron transporters and for amplification purposes.[25–29] An interesting example is the biosensor developed by Liu et al. for the determination of miRNA-21 using an indium tin oxide/polyethylene terephthalate (ITO/PET) electrode modified with a hybrid DNA hydrogel. Ferrocene (Fc)-labeled recognition probes were cross-linked with DNAs grafted onto polyacryla- mide backbones. The hybridization of the target miRNA with the recognition probe led to hydrogel dissolution, the loss of Fc tags, and a reduction in the Fc oxidation current monitored by DPV (Figure 7).[28] Mao et  al. reported the possibility of employing these bio- materials as 3D electron transporters imparting the modified electrodes better electron transfer and signal output intensity compared to the conventional non-functionalized electrodes.[29] The generation of hydrogels on the electrode surface using different strategies has also been exploited for amplification purposes in the development of impedimetric biosensors for the determination of heparanase and Hg2+.[26,25] It is worth noting that the latter method also exploited pH-stimulation of the hydrogel density to achieve signal amplification. 2.3. Multifunctional DNA Probes This section discusses the use of DNA probes capable of per- forming additional functions other than recognition of the target, such as polyA-probes and DNAzymes. 2.3.1. PolyA Probes PolyA-type probes comprising a polyA tail and a recognition part possess interesting capabilities for developing electro- chemical nucleic acid biosensors.[30] This type of probes can strongly adsorb on the gold electrode surface with an affinity comparable to that of the AuS chemical bond.[47] Similarly to the attractive ternary monolayers of thiolated probes pro- posed already a decade ago,[48] PolyA probes allow the control of the capture probe density and spacing to achieve optimal hybridization efficiency, minimize nonspecific adsorptions, and impart antifouling properties to the modified surface.[49] Furthermore, unlike thiol monolayers, PolyA probes are mono- component monolayers with no thiol chemistry involved, exhib- iting enhanced storage stability due to the increased number of the probe anchor points to the gold surface. As can be seen in Table 1, so far, single polyA capture probes (Figure 8a)[30] or multiblock type (two recognition sequences connected through a polyA fragment (Figure 8b)[31] have been employed. It is also important to remark that the use of this type of probes, cheaper than the thiolated ones,[50] allows modulation at will of the size Figure 7. Hybrid DNA hydrogel-based biosensor for the determination of miRNA-21. Reproduced with permission.[28] Copyright 2018, Elsevier. Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (13 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH of the polyA fragments and the evaluation of their influence on the hybridization efficiency and on other properties of the bio- platform such as the storage stability and the antifouling ability. PolyA probes-based electrochemical bioplatforms have been reported to analyze bacterial 16S rRNA,[30] asymmetric PCR amplicons from bacterial genomic DNA,[31] and transgene- derived long RNA.[32] These three bioplatforms involved sandwich hybridization formats assisted by the use of one or multiple reporter probes, enzymatic labeling with HRP and chronoamperometric detection with the 3,3′,5,5′-tetramethylb- enzidine (TMB)/H2O2 system.[30–32] 2.3.2. DNAzymes Signal amplification catalyzed by nanozymes, single-stranded nucleic acids able to catalyze a specific chemical reaction, has been used in the development of electrochemical bioplaforms to achieve the highly sensitive detection of different target ana- lytes: telomerase, Hg2+, miRNA-21, alkaline phosphatase (ALP), carcinoembryonic antigen (CEA), Escherichia coli, and cancer derived exosomes.[3,33–37] The selected strategies are summarized in Table  1. Among them, it is worth mentioning the ratiometric approach reported by Li et  al. to detect Hg2+ involving a bifunctional DNAzyme nanodevice with two detection paths toward the same target.[34] A universal paper-based electrochemical sensor (PES) has been proposed relying on the target-induced synthesis of Mg2+- dependent DNAzyme to catalyze the cleavage of a Fc-labeled probe from paper, thus leading to the release of signal molecules which generated an increased DPV electrochemical signal on CNTs-SPEs (Figure 9a).[35] This strategy claimed zero-background current and allowed the highly sensitive detection of miRNA-21, ALP, and CEA in connection with a miRNA recognition probe, a phosphorylated hairpin probe and a DNA aptamer, respectively. Pandey et  al. reported recently a handheld platform involving the use of electroactive RNA-cleaving DNAzymes (e-RCDs) dually functionalized with biotin and MB and integrated into a two-channel electrical chip with nanostructured electrodes for detecting urinary tract infections in less than 1 h.[36] Figure  9b shows as this strategy implied a differential electrochemical signal readout between two channels, a capture channel where the DPV response of MB increased in the presence of the target bacterium (E. coli) and a release channel where it decreased. Figure 8. a) Single or b) triblock polyA-probes involved in the preparation of electrochemical biosensors for interrogating bacterial 16S rRNA or genomic DNA, respectively. In (a) RR and CR are reporting and capturing region, respectively. (a) Reproduced with permission.[30] Copyright 2019, ACS; (b) Reproduced with permission.[31] Copyright 2019, ACS. Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (14 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH Li´s group has recently reported a paper-based electrochem- ical biosensor to detect cancer-related exosomes that combines the use of a Zr-MOF and an aptamer as recognition elements and HCR and the formation of a DNAzyme for amplification purposes.[37] In this method, the exosomes were absorbed on the surface of Zr-MOFs through the formation of ZrOP bonds. An aptamer capable of specifically recognizing a cancer- derived exosome membrane protein (CD63) initiated the HCR reaction generating double-strands DNA nanowires designed to contain numerous hemin/G-quadruples DNAzymes, which can catalyze the oxidation of TMB, reducing the TMB DPV signal response on the SPE (Figure 9c). 3. Multifunctional Aptamers Aptamers are defined sequences of single-stranded RNA or DNA selected in vitro (systematic evolution of ligands by exponential enrichment) that interact in a tailored 3D design with the target molecule, mirroring antigen–antibody natural Figure 9. a) Universal PES assisted by sequence-specific Mg2+-dependent DNAzymes for determination of different target analytes. b) Portable platform based on the use of e-RCDs and a differential electrochemical signal readout for the determination of Escherichia coli. c) MOF-functionalized paper- based electrochemical biosensor for the determination of exosomes. (a) Reproduced with permission.[35] Copyright 2019, ACS; (b) Reproduced with permission.[36] Copyright 2020, NPG; (c) Reproduced with permission.[37] Copyright 2021, ACS. Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (15 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH interaction. At present, they are gaining a great role in devel- oping competitive electrochemical bioplatforms as evidenced by the recent appearance of several nice reviews and edito- rials.[51–54] Aptamers are advantageous over antibodies in terms of smaller size, chemical stability, rapid, easy, reversible dena- turation capacity, and affordable synthesis and modification, without requiring cell or animal culture and free of batch varia- tions. Moreover, they can be generated against a wide variety of target molecules and selected in deep eutectic solvents in case the target species were poorly soluble in water. To enhance the capabilities they currently provide, there is a tendency to design and exploit multifunctional aptamers. Table 2 summarizes the characteristics of selected recent exam- ples of electrochemical bioplatforms that used polyA-aptameric probes, aptazymes, and dimeric aptamers.[50,55,56] In this context, Wang et  al. reported a simple two-in-one electrochemical biosensor for the simultaneous determination of pesticides and heavy metal ions (Chlorpyrifos and Pb2+ used as models) by designing a dual recognition aptazyme beacon (DRAB).[55] In this approach, the target pesticide activated the self-blocked DRAB by inducing a conformational change. In the presence of the target metal, a MB-tagged signal probe was selectively cleaved resulting in a decrease of the MB electro- chemical response monitored by DPV with the concentration of both analytes. Moreover, the released DRAB-pesticides com- plex can bind adjacent signal probes and start another cyclic cleavage. To improve the sensitivity, multivalent aptamers have been designed by binding an appropriate number of mono- mers. Due to the particular relevance in these days, it should be mentioned the use of dimeric aptamers for the determination of the homotrimeric SARS-CoV-2 spike pro- tein (Figure 10).[56] The resulting aptasensor selectively bound with high affinity the wild-type spike protein and those of vari- ants of concern (Delta first identified in India and Alpha in UK) and was applied to the direct analysis in 1:1 diluted saliva samples from 36 positive and 37 negative patients within 10 min with clinical sensitivity and specificity values of 80.5 and 100%, respectively. 4. Modern Peptides Similarly to aptamers, peptides (amino acid sequences of dif- ferent length and weight comprising synthetic or natural amino acids connected by peptide bonds), are experiencing an unstop- pable rise in developing high performance electrochemical biosensors.[1,6] Peptides are promising affinity biorecognition elements that, in connection with electrochemical transducers, provide advantages primarily in terms of stability, selec- tivity, structural diversity, possibility of tuning the amino acid sequence, and biocompatibility. These recognition elements of synthetic nature can be easily obtained with high yield and functionalized with specific groups (affinity or electroactive tags) for binding (thiol, biotin, etc.) or signaling (Fc, MB, and enzymes) through chemical automated synthesis, thus avoiding the need for laborious in vivo protocols, to enhance binding of Table 2. Selected recently reported multifunctional aptamer-based electrochemical (bio)sensors. Electrode Fundamentals Type of aptamer Detection technique Target analyte/s LR/LOD Sample/ application Remarkable features Ref. SPE Direct affinity reaction PolyA-aptamer DPV (Fe(CN)6]−3/−4) Signal-off Pb2+ 0.1–1000 ng mL−1/0.03 ng mL−1 Spiked river and tap water and fish samples Label-free. Simple and one-step fabrication and 2 min response time [50] AuE Dual-recognition aptazyme beacon and MB-methylene tagged signal probe Aptazyme DPV (MB) Signal-off Chlorpyrifos and Pb2+ Chlorpyrifos: 0.5 nm– 0.5 μM/0.178 nm Pb2+: 0.1 nm– 0.5 μM/0.034 nm Complex food samples Simultaneous determination of targets with different chemical properties [55] AuE Direct affinity reaction Dimeric aptamer EIS (Fe(CN)6]−3/−4) Signal-off Spike proteins of the wildtype, alpha and delta variants of SARS-CoV-2 –/1000 viral particles mL−1 Saliva 10 min assay time. Validation with saliva samples from 73 patients clinical sensitivity of 80.5% and specificity of 100% [56] –: not indicated;. DPV: differential pulse voltammetry; EIS: electrochemical impedance spectroscopy; MB: methylene blue; SPE: screen-printed electrode. Figure 10. Electrochemical biosensor based on a dimeric aptamer for the simple and rapid detection of spike proteins of the wildtype SARS-CoV-2 virus and Alpha and Delta variants. Reproduced with permission.[56] Copyright 2021, Wiley. Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (16 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH peptide in a particular way while retaining its high affinity to the targets.[1,6] Although the advantages of peptides over anti- bodies in the development of electrochemical biosensors are evident as to their smaller size, cost-effectiveness, high yield, and ease of chemical synthesis, their comparison with nucleic acids is not straightforward. Peptides have a different acid–base behavior than nucleic acids and different functional groups that can enhance interactions, and thus affinity, with the target analyte. Peptides have been employed as i) recognition elements; ii) enzyme substrates; iii) electrode modifiers (linker and scaffold), by harnessing their ability to interact and self-assemble to create superior highly ordered nanoscaffolds with good inherent elec- tronic properties and biocompatibility, allowing the immobiliza- tion of other bioreceptors in a given arrangement or imparting antifouling properties to the surface;[57] and iv) labeling/carrier agents for signal amplification.[1] As biorecognition elements they can be exploited to interrogate a wide variety of analytes (nucleic acids, proteins, antibodies, cells, metal ions, etc.) and, as enzyme substrates, play an irreplaceable role to determine the activity (or inhibiting) of clinically relevant enzymes such as proteases (the peptide serves as cleavage-sensing element) and kinases (catalyzed phosphorylation reactions).[1,6] Because of their distinguished properties and the opportunities they impart to the final devices, this section discusses also the use of antifouling, multifunctional, phage-displayed, and aberrant peptides. The characteristics and performance of the selected biosensors are summarized in Table 3. With the aim of developing biosensing devices suitable for real-world applications, peptides that impart antifouling prop- erties to modify platforms, mostly zwitterionic peptides, have been increasingly employed.[57–59,61,63,64,67,68,70,71] In these bio- sensors, the peptide, comprising both a binding domain and an antifouling domain, was used as a bioreceptor[59,61,67,68,71] or as a surface modifier coupled with another bioreceptor (specific aptamers[58,63,64] or DNA probes[57,70]). Another current trend is the use of multifunctional peptides. Among them, one can distinguish between those performing different functions (recognition and amplification)[62] and those more common that have different domains (anchoring, doping, linking, and antifouling), also known as “all-in-one” peptides.[59,63,67,68,71] Importantly, some have been designed with multiple binding domains (multimeric peptides) (Figure 11a)[68] to improve the sensitivity of the determination. In this context, it is noteworthy to mention the strategy developed by Tang et al. to identify stem-like cells in breast tumor using multifunctional peptide-based self-assembled nanofibers that, in addition to the selective analyte recognition, can amplify the response by acting as nanotransporters of multiple AgNPs to generate the electrochemical signal (Figure 11b).[62] It is important to emphasize that some of these multifunc- tional peptides have been designed with characteristics shapes: Y-shaped[57,65] or branched,[68,60] providing advantages for the simplification of the biosensor manufacturing process and out- performing the properties imparted to the resulting biosensor compared to linear peptides.[65,60] Interestingly, the shape of some of these peptides (inverted Y-shaped) has been exploited to prepare scaffolds that, in addition to endowing antifouling properties, improve the sensitivity of the resulting biosensor by ensuring optimal spacing between the immobilized biorecep- tors allowing a more efficient biorecognition (Figure 11c).[57] Phage display technology, which involves the expression of foreign peptides or proteins outside the virion of a phage (a virus that only infects bacteria), has shown to be extremely useful for the production of novel receptors, such as peptides, beyond known biomolecular interactions, or against targets which are toxic or non-immunogenic.[72] Recently, Barderas and Campuzano/Pingarrón research groups reported a bioplatform that used phage display and aberrant peptides for interrogating the potential of their corresponding autoantibodies as reliable biomarkers for diagnosing Alzheimer’s disease (AD) patients. The bioplatform involved the use of MBs modified with the identified four phage-derived and two aberrant HaloTag mono- meric peptides to capture the corresponding serum autoanti- bodies efficiently and selectively.[69] The diagnostic capability of the bioplatforms was assessed with a cohort of 20 serum sam- ples (10 from healthy subjects and 10 from AD patients). The excellent values of area under curve, sensitivity, and specificity, all above 90%, and 100% combining all peptides, obtained by receiver operating characteristic curve analysis, proved the high diagnostic ability of the developed bioplatforms for discrimi- nating AD patients in ≈2 h, in a simple, affordable, and point- of-care manner. 5. CRISPR/Cas Systems CRISPR/Cas systems (clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas)) are surveillance ribonucleoprotein complexes consisting of a single guide RNA with a Cas system such as Cas9, deactivated or mutated Cas9 (dCas9), Cas12a (CPF1), and Cas13a (CPF2), to bind a target sequence (dCas9) or cleave a target DNA/RNA (Cas9, Cas12a, and Cas13a) and generate a signal readout.[73] CRISPR/Cas systems constitute powerful multifunctional biomaterials in contemporary biosensing[74–79] and, in par- ticular, in electrochemical biosensing, improving the detection limits and accuracy for sensing both nucleic acids targets (Cas9: dsDNA; Cas12: ssDNA, and Cas13: ssRNA) or non-nucleic acid targets with high efficiency and simplified designs.[80,81] Due to their selectivity, programmability, and double func- tionality, these systems have been exploited in electrochemical biosensors as: i) biorecognition elements for individual or mul- tiplexed interrogation of nucleic acids (DNAs,[82] miRNAs,[80,83] mRNAs,[84] and viral RNA (Figure 12a)[85]); and ii) signal ampli- fiers for determining non-nucleic acid targets, such as proteins (Figure 12b), metals and bacteria.[86,87] As can be seen in Figure  12a, the strategy developed by Liu et  al. is based on the coupling of CRISPR/Cas technology with electrochemical detection using a personal glucometer (PGM) for COVID-19 screening.[85] In this strategy, a spe- cific gene region of interest (N gene) of SARS-CoV-2 RNA was amplified by RCA. The RCA products bound to Cas12a/ crRNA, thus activating Cas12a which cleaved the polyinvertase- DNA sequences immobilized on magnetic nanoparticles. The released invertase catalyzed the hydrolysis of sucrose to glu- cose, which was detected by the PGM. On the other hand, Chen et al. (Figure 12b) employed the CRISPR/Cas13a system as an Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (17 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH Table 3. Selected recent electrochemical biosensors involving multifunctional peptides. Electrode Fundamentals Type of peptide Detection technique Target analyte/s LR/LOD Sample/ application Remarkable features Ref. Macroporous Au substrate (multilayer polystyrene nanospheres self-assembled on GCE) Direct affinity reaction at an aptamer self-assembled with a zwitterionic peptide onto a macroporous Au substrate Antifouling peptide DPV (Fe(CN)6]−3/−4) Signal-off IgE 0.1–10 pg mL−1/ 42 fg mL−1 Spiked bovine serum solutions Antifouling High sensitivity [58] GCE modified with elec- tropolymerized PANI nanowires arrays Peptide containing anchoring, antifouling, and recognition domains Multifunc- tional peptide (“all-in-one”) DPV (–) Signal-off IgG 1.0 ng mL−1 –10 µg mL−1/ 0.26 ng mL−1 Human serum Antifouling [59] GCE with electrodeposited PANI films Direct affinity reaction at branched zwitterionic peptides Branched peptides DPV (–) Signal-off MUC1 50–106 cells mL−1/20 cells mL−1 MCF-7 cells in human serum Antifouling. 6 order dynamic range. [60] AuNPs/ITO Two antifouling zwitterionic peptides: a longer peptide functionalized with a GO-Fe3O4-Thi probe, which contain the PSA recognition sequence and a shorter one tagged with Fc and used as internal reference Antifouling peptides DPV (Thi) CA (GO-Fe3O4 as peroxidase mimic) + Thi) Signal-off (DPV) Signal-on (CA) PSA 5 pg mL−1–10 ng mL−1/0.76 pg mL−1 (DPV) and 0.42 pg mL−1 (CA) Human serum Dual-mode PSA sensor. Antifouling properties. [61] AuE Capture of BCSCs by a specific aptamer and recognition by MNFs able to recruit AgNPs Multifunctional peptide probes (peptide-based MNFs as nanocar- riers of signaling elements) LSV (AgNO3) BCSCs 10–5 × 105 cells mL−1/6 cells mL−1 Human serum – [62] AuNPs-GCE Direct affinity reaction at an aptamer covalently immobilized onto multifunctional peptides (anchoring, doping, linking, and antifouling sequences) attached to AuNPs-GCE with electropolymerized PEDOT Multifunc- tional peptides (“all-in-one”) DPV (Fe(CN)6]−3/−4) Signal-off miRNA-21 10.0 fm–1.0 nm/2.3 fm Human serum Long-term antifouling performances [63] PABA/GCE Direct affinity reaction at an hairpin aptamer immobilized together with an antifouling peptide onto a PABA/GCE Antifouling peptide DPV (Fe(CN)6]−3/−4) Signal-off IgE 0.001 ng mL−1– 50.0 ng mL−1/0.52 pg mL−1 FBS Antifouling [64] PEDOT-citrate/ AuNPs-GCE Y-shaped peptide with both recognizing and antifouling branches Y-shaped peptides DPV (Fe(CN)6]−3/−4) Signal-off IgG 100 pg mL−1–10 µg mL−1/32 pg mL−1 Human serum Simple fabrication Label-free Antifouling properties [65] AuE Poly adenine coating com- bined with highly specific CD20 epitope mimetic peptide CD20 epitope mimetic peptide EIS (Fe(CN)6]−3/−4) Signal-on Rituximab 0.1–50 µg mL−1/35.26 ng mL−1 Plasma Antifouling [66] PEDOT-citrate film-GCE Direct affinity reaction at a three-in-one peptides Multifunc- tional peptides (all-in-one) DPV (Fe(CN)6]−3/−4) Signal-off APN and human HepG2 cells 1 ng mL−1–15 µg mL−1; 50 to 5 × 105 cells mL−1/0.4 ng mL−1; 20 HepG2 cells mL−1 Human urine Antifouling [67] PANI nanowires-GCE Direct hybridization of the target DNA with biotinylated specific probes immobilized onto inverted Y-shaped peptides-coated PANI nanowires Inverted Y-shaped peptides DPV (Fe(CN)6]−3/−4) Signal-off N-gene (nucleo- capsid phospho- protein) of SARS-CoV-2 10−14–10−9m/3.5 fm Human serum Antifouling High sensitivity [57] Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (18 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH amplification strategy in a sandwich immunoassay.[86] They used a biotinylated double-stranded DNA containing a T7 pro- moter sequence that can be recognized by T7 polymerase to per- form transcription producing many copies of single-stranded RNA molecules. The transcribed RNA molecules activated the CRISPR/Cas13 system which cleaved the reporter DNA. The unprecedented possibilities of these systems, still in their infancy, are encouraging the development of innovative electrochemical biosensor devices achieving extremely high sensitivity (zeptomolar concentrations and single nucleotide resolution of nucleic acids and femtomolar concentrations of non-nucleic acid targets),[87] as well as specificity with simple, fast, and robust performance even on microfluidic devices.[80,83] 6. Opportunities, Challenges, and Perspectives The rational application of nanostructured and/or multi- functional nucleic acid or peptide biomaterials (including the CRISPR/Cas systems) has made possible the birth of a new generation of electrochemical biosensors with unique attributes and capabilities for application. In the last years, nanostructured and multifunctional bioma- terials have demonstrated similar potential to artificial nano- structures for using as electrode modifiers and carriers but lag far behind them in development and application. Simply put, the works critically discussed in this perspective article show that nucleic acid and peptide biomaterials have the Janus side of bioreceptor and nanomaterial. As bioreceptors, they have rel- evant features with respect to other long adopted bioreceptors such as the antibodies in terms of smaller size, chemical sta- bility, rapid, easy, inexpensive and cell and animal-free synthesis and modification, and free of batch variations. As nanomate- rials, they are competitive versus the artificial ones in terms of selectivity, easy tailored structure and shape, lack of variability between batches, lower toxicity compared with carbon nanoma- terials, and not to be bioinert or stiff like metal oxides. For all these reasons, it is impossible not to be convinced that we are at the beginning of a research area on the verge of exponential growth. Although the potential of electrochemical biosensors based on nanostructured and multifunctional nucleic acid and peptide biomaterials is immense, it is important to highlight that these devices should continue collecting medals to earn the recogni- tion of the scientific community and the interdisciplinary sup- port necessary to progress in their advances and developments. Fundamental studies aimed at getting an accurate predic- tion of the supramolecular organization from the biomate- rial sequence and self-assembly mechanisms are required to select the best conditions. It is also imperative to advance in the identification and design of new peptide sequences, so far significantly smaller in number than other receptors such as antibodies, which limits the number of molecular targets that can be analyzed. In addition, novel technologies such as phage Electrode Fundamentals Type of peptide Detection technique Target analyte/s LR/LOD Sample/ application Remarkable features Ref. PEDOT-AuNPs/GCE Direct affinity reaction at a “all-in-one” branched zwit- terionic peptide Multifunctional branched peptide (“all-in-one”) DPV (–) Signal-off IgG 0.1 ng mL−1–10 µg mL−1/45 pg mL−1 Human serum Antifouling Five orders of magnitude [68] SPCE Direct affinity reaction onto MBs modified with the HaloTag peptides Phage-derived and aberrant HaloTag peptides Amperometry Autoanti- bodies – Human serum Identify and validate a new signature of auto- antibodies against the identified pep- tides to diagnose Alzheimer disease patients [69] PANI/GCE Covalent immobilization of a specific DNA probe and an antifouling zwitterionic peptide onto the PANI/GCE Antifouling peptide DPV (–) Signal-off miRNA-24 10 fm– 1.0 nmnm/3.1 fm Human serum Antifouling [70] MXene-Au-MB composite-GCE Direct affinity reaction at a multifunctional peptide (anchoring, antifouling, and recognizing sequences) modified with the signal probe HOOC-MBs anchored to a MXene-Au- MB-modified GCE Multifunc- tional peptide (all-in-one) DPV (Fc and MB) Signal-off (Fc) PSA, TB 5 pg mL−1–10 ng mL−1/0.83 pg mL−1 Human serum Ratiometric Antifouling [71] –: not indicated;. AgNPs: silver nanoparticles; APN: aminopeptidase N; AuE: gold electrode; AuNPs: gold nanoparticles; BCSCs: breast cancer stem cells; CA: chronoamperometry; DPV: differential pulse voltammetry; EIS: electrochemical impedance spectroscopy; FBS: fetal bovine serum; Fc: ferrocene; GCE: glassy carbon electrode; GO: graphene oxide; ITO: indium tin oxide; MB: methylene blue; MBs: magnetic microbeads; MNFs: multifunctional nanofibers; LR: linear range; LOD: limit of detection; LSV: linear square voltammetry; MUC1: mucin 1 protein; PABA: poly (m-aminobenzoic acid); PANI: polyaniline; PEDOT: poly (3,4- ethylenedioxythiophene); PSA: prostate-specific antigen; SPCE: screen-printed carbon electrode; TB: thrombin; Thi: thionine. Table 3. Continued. Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (19 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH display and HaloTag should be used in the peptide production and/or from their expression as fusion proteins, respectively. General requirements and basic performance characteris- tics of the resulting biosensors, such as robustness, precision, specificity, selectivity, stability, etc. must be thoroughly evaluated and their usefulness tested in challenging real-world samples to assure that the capabilities of these biotools are not being overestimated and that it is indeed worth embarking on their further development. In fact, their ability to determine mole- cular targets without complicated sample processing steps, a subject always pending in biosensors, and the antifouling prop- erties imparted by biomaterials, make these biosensors more appropriate than others to address successfully the challenging goals faced. However, it is also important to be aware that the early development stage of these devices and the applications with which they have been confronted may be responsible that Figure 11. Schematic illustration of biosensors prepared using multifunctional peptides a) with a branched shape, b) acting both as detector biore- ceptor and nanocarrier of signaling elements, and c) to construct an antifouling scaffold to immobilize bioreceptors with the appropriate spacing. (a) Reproduced with permission.[68] Copyright 2021, RSC; (b) Reproduced with permission.[62] Copyright 2019, ACS; (c) Reproduced with permission.[57] Copyright 2021, ACS. Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (20 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH the true limitations of these biomaterials have not yet become apparent. Nevertheless, some drawbacks can already be antici- pated. For instance, tetrahedral and Y-shaped nanostructures require the use of multiple probes, are subjected to thiol chem- istry, and restricted to the use of gold surfaces. On the other hand, although the assembly of DNA otrigami is straightforward, the tools to design new structures are not advanced and cannot be truly formalized until the relationships between design and properties are fully understood. As for dendrimers, their stability in physiological matrices needs to be further investigated. It is also expected that the demand for improved capabilities of this biodevices will only increase as they move forward, as more is always demanded to the best, like the urgently needed simultaneous determination of multiple targets, which provides notable advantages such as increased precision, low sample consumption, high sample throughput, and reduced cost and time per assay to avoid misdiagnoses. A great effort is also fore- seen on the development of new amplification strategies and in the design and application of multimeric biomaterials to meet the demand for increasingly sensitive tools to detect analytes at ultra-low levels. Moreover, although these devices have promising potential usages, we must be aware that they are still at the conceptual level. So, their natural development must first go through Figure 12. Strategies based on the use of CRISPR/Cas systems a) as bioreceptors or b) for amplification in electrochemical biosensing to interrogate viral RNA and protein targets, respectively. (a) Reproduced with permission.[85] Copyright 2021, RSC; (b) Reproduced with permission.[86] Copyright 2020, ACS. Adv. Mater. Technol. 2022, 2200310 www.advancedsciencenews.com www.advmattechnol.de 2200310 (21 of 23) © 2022 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH their translation from these proof-of-concepts to real practical devices and then from bench to POC. It is unquestionable that during these challenging transitions many complex obstacles must be overcome which go through simplifying and mini- mizing the fabrication cost and assay time, further improving the sensitivity, selectivity, storage stability, and anti-inter- ference properties of these biodevices, demonstrating their robustness and reliability with a large number of samples and with different users in different environments, and cou- pling them with simple and portable electrochemical detection devices. Further applications will require fresh breakthroughs which skillfully combine the expertise of multidisciplinary researchers. This interdisciplinary research with biomolecular nanomaterials poses great challenges and opportunities to drive the development of high-performance electrochemical biosensors, which can only be achieved with the approval and momentum of the intersecting areas of nanoscience, materials science, and molecular biotechnology. It is important to keep in mind that although both bio and artificial nanomaterials can be used as electrode modifiers, car- riers, or labels, the possibilities they provide are different but not incompatible. We personally believe that the combination of both types of nanomaterials, scarcely exploited to date, can open a new avenue where both the natural and the artificial nanomaterials will be winning horses. A good example of this is the use of DNA networks involved in the “dispersible elec- trodes.” It is clear, for example, that biomaterials can impart to the artificial ones with selectivity which is always highlighted as the weak point of the latter. The same can be said about the use of nanostructured biomaterials combined with other types of more conventional bioreceptors. We think that the delay in the appearance on the scene of nanostructured and multifunctional biomaterials with respect to artificial ones is partly due to a lack of knowledge or to the mistaken thoughts that they are less stable or that their manipulation requires much more advanced knowledge than for artificial nanomaterials. Hopefully, this perspective article will fulfill our purpose of reflecting the simplicity and tremen- dous opportunities of these biomaterials and their competi- tive advantages over other commonly used bioreceptors (such as the tremendously popular antibodies these days for antigen tests to detect COVID-19) or artificial nanomaterials, giving the scientific community the confidence and driving to join efforts and enthusiasm in their exploration. Acknowledgements The financial support of PID2019-103899RB-I00 (Spanish Ministerio de Ciencia e Innovación) Research Project, the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349), and PI20CIII/00019 Grant from the AES-ISCIII program are gratefully acknowledged. Conflict of Interest The authors declare no conflict of interest. Author Contributions Writing, review, and editing: S.C., M.P., R.B., and J.M.P. Funding acquisition: S.C. and R.B. Keywords antifouling, empowered electrochemical biosensors, nanostructured and/or multifunctional biomaterials, nucleic acids, peptides Received: February 27, 2022 Revised: April 7, 2022 Published online: [1] L. Yuan, L. Liu, Sens. Actuators, B 2021, 344, 130232. [2] H. Pei, X. Zuo, D. Pan, J. Shi, Q. Huang, C. Fan, NPG Asia Mater. 2013, 5, e51. [3] J.  Lu, J.  Wang, X.  Hu, E.  Gyimah, S.  Yakubu, K.  Wang, X.  Wu, Z. Zhang, Anal. Chem. 2019, 91, 7353. 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Her areas of interest include the development of affinity-based electrochemical bioplatforms with potential for multiplexed and/or multi-omics determinations in clinical and food safety. She is associate editor of the journal Electroanalysis (Wiley-VCH). María Pedrero is associate professor in the Analytical Chemistry Department of the Chemistry Faculty at the Universidad Complutense de Madrid (Spain) since 2002. She collaborates in the “Electroanalysis and Electrochemical (Bio)sensors” (GEBE) research group. Her main areas of interest include the development of enzymatic, immune, and DNA electrochemical sensors, at present for the detection of proteins and oligonucleotides as markers of cancer, cardiovascular, and neurological diseases. Rodrigo Barderas received his Ph.D. in Chemistry (Biochemistry and Molecular Biology) from the Complutense University of Madrid (Spain) in 2004. Since 2004 he has been working in the field of proteomics and high-throughput screening techniques. In 2017 got a Tenured Scientist position at the Instituto de Salud Carlos III. He is currently the Head of the Functional Proteomics Unit of the Chronic Disease Programme. His areas of interest include the identification of diagnostic, and prognostic markers and new targets of intervention in chronic diseases of high prevalence by using proteomics and phage display, particularly in colorectal cancer. José M. Pingarrón is professor of Analytical Chemistry and member of the group “Electroanalysis and electrochemical(bio)sensors” at Complutense University of Madrid. His research lines include the development of nanostructured electrochemical platforms (enzyme, immuno-, and genosensors) for single or multiplexed determination of relevant biomarkers. He is Senior Honorary Advisor of the journal Electroanalysis and since 2017 Fellow of the International Society of Electrochemistry. Currently he holds the position of Secretary General of Universities in the Spanish Government. Adv. Mater. Technol. 2022, 2200310