Breaking barriers in electrochemical biosensing using bioinspired peptide and phage probes.

Electrochemical biosensing continues to advance tirelessly, overcoming barriers that have kept it from leaving research laboratories for many years. Among them, its compromised performance in complex biological matrices due to fouling or receptor stability issues, the limitations in determining toxic and small analytes, and its use, conditioned to the commercial availability of commercial receptors and the exploration of natural molecular interactions, deserved to be highlighted. To address these challenges, in addition to the intrinsic properties of electrochemical biosensing, its coupling with biomimetic materials has played a fundamental role, among which bioinspired phage and peptide probes stand out. The versatility in design and employment of these probes has opened an unimaginable plethora of possibilities for electrochemical biosensing, improving their performance far beyond the development of highly sensitive and selective devices. The state of the art offers robust electroanalytical biotools, capable of operating in complex samples and with exciting opportunities to discover and determine targets regardless of their toxicity and size, the commercial availability of bioreceptors, and prior knowledge of molecular interactions. With all this in mind, this review offers a panoramic, novel, and updated vision of both the tremendous advances and opportunities offered by the combination of electrochemical biosensors with bioinspired phage and peptide probes and the challenges and research efforts that are envisioned in the immediate future.

Electrochemical bioplatform to manage alpha-gal syndrome by tracking the carbohydrate allergen in meat.

This work presents the first bioplatform described to date for the determination of galactose-α-1,3-galactose (α-Gal), a non-primate mammalian oligosaccharide responsible for almost all cases of red meat allergy. The bioplatform is based on the implementation of an indirect competitive immunoassay and enzymatic labeling with the enzyme horseradish peroxidase (HRP) built on the surface of magnetic microparticles (MBs) and amperometric transduction on screen-printed carbon electrodes (SPCEs) using the H2O2/hydroquinone (HQ) system. The target α-Gal competed with biotinylated α-Gal immobilized on the surface of neutravidin-modified MBs for the limited immunorecognition sites of a detection antibody enzymatically labeled with an HRP-conjugated secondary antibody. The resulting magnetic immunoconjugates were trapped on the surface of the SPCE working electrode and amperometric transduction was performed, providing a cathodic current variation inversely proportional to the concentration of α-Gal in the analyzed sample. The developed biotool was optimized, characterized and applied with satisfactory results to the determination of the target allergen in different samples of raw and processed meats.

Bringing to Light the Importance of the miRNA Methylome in Colorectal Cancer Prognosis Through Electrochemical Bioplatforms.

This work reports the first electrochemical bioplatforms developed for the determination of the total contents of either target miRNA or methylated target miRNA. The bioplatforms are based on the hybridization of the target miRNA with a synthetic biotinylated DNA probe, the capture of the formed DNA/miRNA heterohybrids on the surface of magnetic microcarriers, and their recognition with an antibody selective to these heterohybrids or to the N6-methyladenosine (m6A) epimark. The determination of the total or methylated target miRNA was accomplished by labeling such secondary antibodies with the horseradish peroxidase (HRP) enzyme. In both cases, amperometric transduction was performed on the surface of disposable electrodes after capturing the resulting HRP-tagged magnetic bioconjugates. Because of their increasing relevance in colorectal cancer (CRC) diagnosis and prognosis, miRNA let-7a and m6A methylation were selected. The proposed electrochemical bioplatforms showed attractive analytical and operational characteristics for the determination of the total and m6A-methylated target miRNA in less than 75 min. These bioplatforms, innovative in design and application, were applied to the analysis of total RNA samples extracted from cultured cancer cells with different metastatic profiles and from paired healthy and tumor tissues of patients diagnosed with CRC at different stages. The obtained results demonstrated, for the first time using electrochemical platforms, the potential of interrogating the target miRNA methylation level to discriminate the metastatic capacities of cancer cells and to identify tumor tissues and, in a pioneering way, the potential of the m6A methylation in miRNA let-7a to serve as a prognostic biomarker for CRC.

Micromotor-based electrochemical immunoassays for reliable determination of amyloid-ß (1-42) in Alzheimer's diagnosed clinical samples.

Alzheimer's disease (AD), in addition to being the most common cause of dementia, is very difficult to diagnose, with the 42-amino acid form of Aß (Aß-42) being one of the main biomarkers used for this purpose. Despite the enormous efforts made in recent years, the technologies available to determine Aß-42 in human samples require sophisticated instrumentation, present high complexity, are sample and time-consuming, and are costly, highlighting the urgent need not only to develop new tools to overcome these limitations but to provide an early detection and treatment window for AD, which is a top-challenge. In recent years, micromotor (MM) technology has proven to add a new dimension to clinical biosensing, enabling ultrasensitive detections in short times and microscale environments. To this end, here an electrochemical immunoassay based on polypyrrole (PPy)/nickel (Ni)/platinum nanoparticles (PtNPs) MM is proposed in a pioneering manner for the determination of Aß-42 in left prefrontal cortex brain tissue, cerebrospinal fluid, and plasma samples from patients with AD. MM combines the high binding capacity of their immunorecognition external layer with self-propulsion through the catalytic generation of oxygen bubbles in the internal layer due to decomposition of hydrogen peroxide as fuel, allowing rapid bio-detection (15 min) of Aß-42 with excellent selectivity and sensitivity (LOD = 0.06 ng/mL). The application of this disruptive technology to the analysis of just 25 µL of the three types of clinical samples provides values concordant with the clinical values reported, thus confirming the potential of the MM approach to assist in the reliable, simple, fast, and affordable diagnosis of AD by determining Aß-42.

Redox Cycling Amplified Electrochemical Lateral-Flow Immunoassay: Toward Decentralized Sensitive Insulin Detection.

While paper-based lateral-flow immunoassays (LFA) offer considerable promise for centralized diagnostic applications, the analytical capability of conventional LFA remains constrained due to the low sensitivity of its common optical detection strategy. To address these issues, we report a simple electrochemical LFA (eLFA) with nanocatalytic redox cycling for decentralized insulin detection. Simultaneous binding of insulin with detection antibodies and capture antibodies through the capillary flow at the LFA platform and signal amplification through the rapid nanocatalytic reduction of [Fe(CN)6]3- (Fe3+) with Au nanoparticles (AuNP) and ammonia-borane (AB), coupled to electrochemical redox cycling reactions involving Fe3+, AuNP, and AB on the carbon working electrode, offer higher sensitivity than conventional colorimetric LFA and enzymatic redox cycling. The resulting integrated eLFA strip allows the detection of low insulin concentrations (LOD = 12 pM) and offers considerable promise for highly sensitive decentralized assays of different biological fluids (saliva and serum) without additional pretreatment or washing steps.

Electrochemical Affinity Biosensors: Pervasive Devices with Exciting Alliances and Horizons Ahead.

Electrochemical affinity biosensors are evolving at breakneck speed, strengthening and colonizing more and more niches and drawing unimaginable roadmaps that increasingly make them protagonists of our daily lives. They achieve this by combining their intrinsic attributes with those acquired by leveraging the significant advances that occurred in (nano)materials technology, bio(nano)materials and nature-inspired receptors, gene editing and amplification technologies, and signal detection and processing techniques. The aim of this Perspective is to provide, with the support of recent representative and illustrative literature, an updated and critical view of the repertoire of opportunities, innovations, and applications offered by electrochemical affinity biosensors fueled by the key alliances indicated. In addition, the imminent challenges that these biodevices must face and the new directions in which they are envisioned as key players are discussed.

Electrochemical biotool for the dual determination of epithelial mucins associated to prognosis and minimal residual disease in colorectal cancer.

This work reports a dual immunoplatform for the simultaneous detection of two epithelial glycoproteins of the mucin family, mucin 1 (MUC1) and mucin 16 (MUC16), whose expression is related to adverse prognosis and minimal residual disease (MRD) in colorectal cancer (CRC). The developed immunoplatform involves functionalised magnetic microparticles (MBs), a set of specific antibody pairs (a capture antibody, cAb, and a biotinylated detector antibody b-dAb labelled with a streptavidin-horseradish peroxidase, Strep-HRP, polymer) for each target protein and amperometric detection at dual screen-printed carbon electrodes (SPdCEs) using the hydroquinone (HQ)/horseradish peroxidase (HRP)/H2O2 system. This dual immunoplatform allows, under the optimised experimental conditions, to achieve LOD values of 50 and 1.81 pg mL-1 (or mU mL-1) for MUC1 and MUC16, respectively, and adequate selectivity for the determination of the two targets in the clinic. The developed immunoplatform was employed to analyse CRC cell protein extracts (1.0 µg/determination) with different metastatic potential providing results in agreement with those obtained by blotting technologies but using affordable and applicable point-of-care instruments. This new biotool also emerges competitive in state-of-the-art electrochemical immunoplatforms seeking a compromise among simplicity, reduction of test time and analytical characteristics.

Pursuing precision in medicine and nutrition: the rise of electrochemical biosensing at the molecular level.

In the era that we seek personalization in material things, it is becoming increasingly clear that the individualized management of medicine and nutrition plays a key role in life expectancy and quality of life, allowing participation to some extent in our welfare and the use of societal resources in a rationale and equitable way. The implementation of precision medicine and nutrition are highly complex challenges which depend on the development of new technologies able to meet important requirements in terms of cost, simplicity, and versatility, and to determine both individually and simultaneously, almost in real time and with the required sensitivity and reliability, molecular markers of different omics levels in biofluids extracted, secreted (either naturally or stimulated), or circulating in the body. Relying on representative and pioneering examples, this review article critically discusses recent advances driving the position of electrochemical bioplatforms as one of the winning horses for the implementation of suitable tools for advanced diagnostics, therapy, and precision nutrition. In addition to a critical overview of the state of the art, including groundbreaking applications and challenges ahead, the article concludes with a personal vision of the imminent roadmap.

Electrochemical bioplatform for the determination of the most common and carcinogenic human papillomavirus DNA.

Nucleic acid-based analytical bioplatforms have gained importance as diagnostic tests for genomics and as early detection tools for diseases such as cancer. In this context, we report the development of an amperometric bioplatform for the determination of a specific human papillomavirus type 16 (HPV16) sequence. The bioplatform utilizes an immune-nucleic acid hybrid-sandwich assay. A biotinylated RNA capture probe (RNAbCp), complementary to the selected HPV16 target DNA sequence, was immobilised on the surface of streptavidin coated magnetic microbeads (Strep-MBs). The RNA/DNA heteroduplex resulting from the hybridization of the RNAbCP and the HPV16 target sequence was recognised by a commercial antibody that specifically bound to the heteroduplex (AbDNA-RNA). A horseradish-peroxide labeled secondary antibody (antiIgG-HRP) was used for the detection of AbDNA-RNA. Relying on amperometric detection of the resulting HRP-labeled magnetic bioconjugates captured on screen-printed electrodes (SPCEs) in the presence of H2O2 and hydroquinone (HQ), the biotool achieved a low limit of detection (0.5 pM) for the synthetic HPV16 target DNA. In addition, the developed bioplatform was able to discriminate between HPV16 positive and negative human cancer cells using only 25 ng of amplified DNA in a test time of 45 min.

Anti-double stranded DNA antibodies: Electrochemical isotyping in autoimmune and neurological diseases.

This work reports the first amperometric biosensor for the simultaneous determination of the single or total content of the most relevant human immunoglobulin isotypes (hIgs) of anti-dsDNA antibodies, dsDNA-hIgG, dsDNA-hIgM, dsDNA-hIgA and dsDNA-three hIgs, which are considered relevant biomarkers in prevalent autoimmune diseases such as systemic lupus erythematosus (SLE) as well as of interest in neurodegenerative diseases such as Alzheimer's disease (AD). The bioplatform involves the use of neutravidin-functionalized magnetic microparticles (NA-MBs) modified with a laboratory-prepared biotinylated human double-stranded DNA (b-dsDNA) for the efficient capture of specific autoantibodies that are enzymatically labeled with horseradish peroxidase (HRP) enzyme using specific secondary antibodies for each isotype or a mixture of secondary antibodies for the total content of the three isotypes. Transduction was performed by amperometry (-0.20 V vs. the Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system after trapping the resulting magnetic bioconjugates on each of the four working electrodes of a disposable quadruple transduction platform (SP4CEs). The bioplatform demonstrated attractive operational characteristics for clinical application and was employed to determine the individual or total hIgs classes in serum from healthy individuals and from patients diagnosed with SLE and AD. The target concentrations in AD patients are provided for the first time in this work. In addition, the results for SLE patients and control individuals agree with those obtained by applying ELISA tests as well as with the clinical ranges reported by other authors, using individual detection methodologies restricted to centralized settings or clinical laboratories.

p53 and p63 Proteoforms Derived from Alternative Splicing Possess Differential Seroreactivity in Colorectal Cancer with Distinct Diagnostic Ability from the Canonical Proteins.

Colorectal cancer (CRC) is the third most common cancer and the second most frequent cause of cancer-related death worldwide. The detection in plasma samples of autoantibodies against specific tumor-associated antigens has been demonstrated to be useful for the early diagnosis of CRC by liquid biopsy. However, new studies related to the humoral immune response in cancer are needed to enable blood-based diagnosis of the disease. Here, our aim was to characterize the humoral immune response associated with the different p53 and p63 proteoforms derived from alternative splicing and previously described as aberrantly expressed in CRC. Thus, here we investigated the diagnostic ability of the twelve p53 proteoforms and the eight p63 proteoforms described to date, and their specific N-terminal and C-terminal end peptides, by means of luminescence HaloTag beads immunoassays. Full-length proteoforms or specific peptides were cloned as HaloTag fusion proteins and their seroreactivity analyzed using plasma from CRC patients at stages I-IV (n = 31), individuals with premalignant lesions (n = 31), and healthy individuals (n = 48). p53γ, Δ40p53ß, Δ40p53γ, Δ133p53γ, Δ160p53γ, TAp63α, TAp63δ, ΔNp63α, and ΔNp63δ, together with the specific C-terminal end α and δ p63 peptides, were found to be more seroreactive against plasma from CRC patients and/or individuals with premalignant lesions than from healthy individuals. In addition, ROC (receiver operating characteristic) curves revealed a high diagnostic ability of those p53 and p63 proteoforms to detect CRC and premalignant individuals (AUC higher than 85%). Finally, electrochemical biosensing platforms were employed in POC-like devices to investigate their usefulness for CRC detection using selected p53 and p63 proteoforms. Our results demonstrate not only the potential of these biosensors for the simultaneous analysis of proteoforms' seroreactivity, but also their convenience and versatility for the clinical detection of CRC by liquid biopsy. In conclusion, we here show that p53 and p63 proteoforms possess differential seroreactivity in CRC patients in comparison to controls, distinctive from canonical proteins, which should improve the diagnostic panels for obtaining a blood-based biomarker signature for CRC detection.

Electrochemical (Bio)Sensing Devices for Human-Microbiome-Related Biomarkers.

The study of the human microbiome is a multidisciplinary area ranging from the field of technology to that of personalized medicine. The possibility of using microbiota biomarkers to improve the diagnosis and monitoring of diseases (e.g., cancer), health conditions (e.g., obesity) or relevant processes (e.g., aging) has raised great expectations, also in the field of bioelectroanalytical chemistry. The well-known advantages of electrochemical biosensors-high sensitivity, fast response, and the possibility of miniaturization, together with the potential for new nanomaterials to improve their design and performance-position them as unique tools to provide a better understanding of the entities of the human microbiome and raise the prospect of huge and important developments in the coming years. This review article compiles recent applications of electrochemical (bio)sensors for monitoring microbial metabolites and disease biomarkers related to different types of human microbiome, with a special focus on the gastrointestinal microbiome. Examples of electrochemical devices applied to real samples are critically discussed, as well as challenges to be faced and where future developments are expected to go.

First PCR-free electrochemical bioplatform for the detection of mustard Sin a 1 protein as a potential "hidden" food allergen.

A disposable electrochemical PCR-free biosensor for the selective detection of a fragment encoding the protein Sin a 1, a 2S albumin considered a diagnostic marker for sensitization to mustard, is reported. The methodology is based on the formation of DNA/RNA heterohybrids by sandwich hybridization of a specific fragment of the Sin a 1 allergen coding sequence with appropriately designed RNA probes. Labeling with commercial antibodies specific to the heteroduplexes and secondary antibodies conjugated with horseradish peroxidase (HRP) was carried out onto the surface of magnetic beads (MBs). Amperometric transduction was undertaken on screen-printed electrodes using H2O2 as enzyme substrate and hydroquinone (HQ) a redox mediator. The electrochemical biosensor allows the simple and fast detection (75 min) of Sin a 1 reaching a limit of detection of 3 pM. The bioplatform was successfully applied to the analysis of the targeted Sin a 1 gene specific region using just 50 ng of non-fragmented denatured genomic DNA extracted from yellow mustard seeds.

First bioelectronic immunoplatform for quantitative secretomic analysis of total and metastasis-driven glycosylated haptoglobin.

The glycosylation status of proteins is increasingly used as biomarker to improve the reliability in the diagnosis and prognosis of diseases as relevant as cancer. This feeds the need for tools that allow its simple and reliable analysis and are compatible with applicability in the clinic. With this objective in mind, this work reports the first bioelectronic immunoplatforms described to date for the determination of glycosylated haptoglobin (Hp) and the simultaneous determination of total and glycosylated Hp. The bioelectronic immunoplatform is based on the implementation of non-competitive bioassays using two different antibodies or an antibody and a lectin on the surface of commercial magnetic microcarriers. The resulting bioconjugates are labeled with the horseradish peroxidase (HRP) enzyme, and after their magnetic capture on disposable electroplatforms, the amperometric transduction using the H2O2/hydroquinone (HQ) system allows the single or multiple detection. The developed immunoplatform achieves limits of detection (LODs) of 0.07 and 0.46 ng mL-1 for total and glycosylated Hp in buffer solution, respectively. The immunoplatform allows accurate determination using simple and relatively short protocols (approx. 75 min) of total and glycosylated Hp in the secretomes of in vitro-cultured colorectal cancer (CRC) cells with different metastatic potentials, which is not feasible, due to lack of sensitivity, by means of some commercial ELISA kits and Western blot methodology.

Multipurpose E-bioplatform targeting Kv channels in whole cancer cells and evaluating of their potential therapeutics.

Potassium ion channels are expressed on the cell membranes, implicated in wide variety of cell functions and intimately linked to cancer cell behaviors. This work reports the first bioplatform described to date allowing simple and rapid detection of ion channel activity and the effect of their inhibitors in cancer cells. The methodology involves interrogation of the channel of interest from cells specifically captured on magnetic immunoconjugates using specific detection antibodies that are labeled with horseradish peroxidase enzyme. The channel activity is reflected by an amperometric signal transduction of the resulting magnetic bioconjugates onto screen-printed carbon electrodes. The bioplatform feasibility was proven for the detection of the Kv channels in U87 human glioblastoma cells and their blocking by scorpion venom KAaH1 and KAaH2 peptides. The obtained results confirm the high sensitivity (detection of 5 U87 cells⋅mL-1 and 0.06 µg mL-1 of KAaH2) of the proposed bioplatform and their versatility to detect both potassium channel activity and their potential inhibitors, in a given cancer cell line, with high sensitivity in a simple and fast way. This bioplatform presents potential applications in cancer and theranostic of channelopathies.

Towards Control and Oversight of SARS-CoV-2 Diagnosis and Monitoring through Multiplexed Quantitative Electroanalytical Immune Response Biosensors.

The development of versatile and sensitive biotools to quantify specific SARS-CoV-2 immunoglobulins in SARS-CoV-2 infected and non-infected individuals, built on the surface of magnetic microbeads functionalized with nucleocapsid (N) and in-house expressed recombinant spike (S) proteins is reported. Amperometric interrogation of captured N- and S-specific circulating total or individual immunoglobulin (Ig) isotypes (IgG, IgM, and IgA), subsequently labelled with HRP-conjugated secondary antibodies, was performed at disposable single or multiplexed (8×) screen-printed electrodes using the HQ/HRP/H2O2 system. The obtained results using N and in-house expressed S ectodomains of five SARS-CoV-2 variants of concern (including the latest Delta and Omicron) allow identification of vulnerable populations from those with natural or acquired immunity, monitoring of infection, evaluation of vaccine efficiency, and even identification of the variant responsible for the infection.

Towards Control and Oversight of SARS-CoV-2 Diagnosis and Monitoring through Multiplexed Quantitative Electroanalytical Immune Response Biosensors.

The development of versatile and sensitive biotools to quantify specific SARS-CoV-2 immunoglobulins in SARS-CoV-2 infected and non-infected individuals, built on the surface of magnetic microbeads functionalized with nucleocapsid (N) and in-house expressed recombinant spike (S) proteins is reported. Amperometric interrogation of captured N- and S-specific circulating total or individual immunoglobulin (Ig) isotypes (IgG, IgM, and IgA), subsequently labelled with HRP-conjugated secondary antibodies, was performed at disposable single or multiplexed (8×) screen-printed electrodes using the HQ/HRP/H2 O2 system. The obtained results using N and in-house expressed S ectodomains of five SARS-CoV-2 variants of concern (including the latest Delta and Omicron) allow identification of vulnerable populations from those with natural or acquired immunity, monitoring of infection, evaluation of vaccine efficiency, and even identification of the variant responsible for the infection.

Dextran-coated nanoparticles as immunosensing platforms: Consideration of polyaldehyde density, nanoparticle size and functionality.

Magnetic nanoparticles (MNPs) can be used as antibody carriers in a wide range of immunosensing applications. The conjugation chemistry for preparing antibody-MNP bionanohybrids should assure the nanoparticle's colloidal dispersity, directional conformation and high biofunctionality retention of attached antibodies. In this work, peroxidase (HRP) was selected as model target analyte, and stable antibody-MNP conjugates were prepared using polyaldehyde-dextrans as multivalent linkers, also to prevent nanoparticles agglomeration and steric shielding of non-specific proteins. Under the manipulation of the oxidation variables, MNP-conjugated antibody showed the highest Fab accessibility, of 1.32 µmol analyte per µmol antibody, corresponding to 139 µmol aldehyde per gram of nanocarrier (5 mM NaIO4, 4 h). Demonstrating anti-interference advantage up to 10% serum, colorimetric immunoassay gave a detection limit (LOD) of 300 ng mL-1, while electrochemical transduction led to a considerable (680 times) improvement, with a LOD of 0.44 ng mL-1. In addition, polyaldehyde-dextran showed priority over polycarboxylated-dextran as the multivalent antibody crosslinker for MNPs in terms of sensitivity and LOD value, while immunosensors constructed with carboxylated magnetic microbeads (HOOC-MBs) outperformed MNPs-based immunoplatforms. This work sheds light on the importance of surface chemistry (type and density of functional groups) and the dimension (nanosize vs micrometer) of magnetic carriers to conjugate antibodies with better directional orientation and improve the analytical performance of the resulting immunosensors.