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.

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.

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 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.

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 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.

Binary MoS2 nanostructures as nanocarriers for amplification in multiplexed electrochemical immunosensing: simultaneous determination of B cell activation factor and proliferation-induced signal immunity-related cytokines.

A dual immunosensor is reported for the simultaneous determination of two important immunity-related cytokines: BAFF (B cell activation factor) and APRIL (a proliferation-induced signal). Sandwich-type immunoassays with specific antibodies (cAbs) and a strategy for signal amplification based on labelling the detection antibodies (dAbs) with binary MoS2/MWCNTs nanostructures and using horseradish peroxidase (HRP) were implemented. Amperometric detection was carried out at screen-printed dual carbon electrodes (SPdCEs) through the hydroquinone HQ/H2O2 system. The developed dual immunosensor provided limit of detection (LOD) of 0.08 and 0.06 ng mL-1 for BAFF and APRIL, respectively, and proved to be useful for the determination of both cytokines in cancer cell lysates and serum samples from patients diagnosed with autoimmune diseases and cancer. The obtained results agreed with those found using ELISA methodologies.

Anticipating metastasis through electrochemical immunosensing of tumor hypoxia biomarkers.

Metastasis is responsible for about 90% of cancer-associated deaths. In the context of solid tumors, the low oxygen concentration in the tumor microenvironment (hypoxia) is one of the key factors contributing to metastasis. Tumor cells adapt to these conditions by overexpressing certain proteins such as programmed death ligand 1 (PD-L1) and hypoxia-inducible factor 1 alpha (HIF-1α). However, the determination of these tumor hypoxia markers that can be used to follow-up tumor progression and improve the efficiency of therapies has been scarcely addressed using electrochemical biosensors. In this work, we report the first electrochemical bioplatform for the determination of PD-L1 as well as the first one allowing its simultaneous determination with HIF-1α. The target proteins were captured and enzymatically labeled on magnetic microbeads and amperometric detection was undertaken on the surface of screen-printed dual carbon electrodes using the hydrogen peroxide/peroxidase/hydroquinone system. Sandwich immunoassays were implemented for both the HIF-1α and PD-L1 sensors and the analytical characteristics were evaluated providing LOD values of 86 and 279 pg mL-1 for the amperometric determination of PD-L1 and HIF-1α standards, respectively. The developed electrochemical immunoplatforms are competitive versus the only electrochemical immunosensor reported for the determination of HIF-1α and the "gold standard" ELISA methodology for the single determination of both proteins in terms of assay time, compatibility with the simultaneous determination of both proteins making their use suitable for untrained users at the point of attention. The dual amperometric immunosensor was applied to the simultaneous determination of HIF-1α and PD-L1 in cancer cell lysates. The analyses lasted only 2 h and just 0.5 µg of the sample was required.

Electrochemical LAMP-based assay for detection of RNA biomarkers in prostate cancer.

Current molecular diagnostics of prostate cancer relies on detection of elevated levels of PSA protein in serum, but its specificity has been questioned due to its higher levels also in non-malignant prostate diseases. A long non-coding RNA biomarker, PCA3, demonstrated excellent specificity for prostate cancer, and thus has become an interesting alternative to PSA monitoring. Its detection utilizes mostly reverse transcription PCR with optical detection, making the protocol longer and more expensive. To avoid PCR, we have developed an electrochemical assay coupled with LAMP, an isothermal amplification technique showing high sensitivities at constant temperatures and shorter reaction times. We amplified PCA3 RNA as well as PSA mRNA (serving as a control), hybridized LAMP products on magnetic beads and measured them with chronoamperometry at carbon electrode chips. We show good sensitivity and specificity for both biomarkers in prostate cancer cell lines, and successful detection of PCA3 in clinical samples, i.e., urine samples from 11 prostate cancer patients and 7 healthy controls, where we obtained excellent correlation with clinical data. This is to our knowledge a first such attempt to apply electrochemistry to determine two RNA biomarkers directly in urine samples of prostate cancer patients in a minimally invasive diagnostics format.

Multiplexed magnetic beads-assisted amperometric bioplatforms for global detection of methylations in nucleic acids.

This work reports the first electrochemical bioplatform developed for the multidetection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in DNA, DNA N6-methyladenine (6mA) and RNA N6-methyladenosine (m6A) methylations at global level. Direct competitive immunoassays were implemented on the surface of magnetic beads (MBs) and optimized for the single amperometric determination of different targets varying in length, sequence and number of methylations on screen-printed carbon electrodes. After evaluating the sensitivity and selectivity of such determinations and the confirmation of no cross-reactivity, a multiplexed disposable platform allowing the simultaneous determination of the mentioned four methylation events in only 45 min has been prepared. The multiplexed bioplatform was successfully applied to the determination of m6A in cellular total RNA and of 5-mC, 5-hmC and 6mA in genomic DNA extracted from tissues. The developed bioplatform showed its usefulness to discriminate the aggressiveness of cancerous cells and between healthy and tumor tissues of colorectal cancer patients.

Simultaneous determination of four fertility-related hormones in saliva using disposable multiplexed immunoplatforms coupled to a custom-designed and field-portable potentiostat.

This work reports the first amperometric immunosensor for the simultaneous determination of four fertility-related hormones in saliva: progesterone (P4), luteinizing hormone (LH), estradiol (E2), and prolactin (PRL). The immune platform involves direct competitive (P4 and E2), and sandwich (LH and PRL) assays implemented onto functionalized magnetic microbeads (MBs). The amperometric transduction was performed upon placing the MBs-immunoconjugates onto each of the four working electrodes of the SPCE array (SP4CEs) and applying a detection potential of -0.20 V (vs. Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system. The achieved analytical and operational characteristics of the developed multiplexed immunoplatform showed a sensitivity that allows the determination of these hormones in saliva, and an adequate selectivity to analyse complex clinical samples. The bioplatform was employed for the determination of the set of four hormones in human saliva samples collected from individuals with different hormonal profiles. The results obtained using a conventional potentiostat were compared with those provided employing a novel low-cost custom-designed and field-portable quadruple potentiostat. Similar results were found which also agreed with those obtained by applying ELISA methods for the determination of single hormones.

Electrochemical Immunosensing of ST2: A Checkpoint Target in Cancer Diseases.

A magnetic beads (MB)-involved amperometric immunosensor for the determination of ST2, a member of the IL1 receptor family, is reported in this work. The method utilizes a sandwich immunoassay and disposable screen-printed carbon electrodes (SPCEs). Magnetic immunoconjugates built on the surface of carboxylic acid-microsized magnetic particles (HOOC-MBs) were used to selectively capture ST2. A biotinylated secondary antibody further conjugated with a streptavidin peroxidase conjugate (Strep-HRP) was used to accomplish the sandwiching of the target protein. The immune platform exhibits great selectivity and a low limit of detection (39.6 pg mL-1) for ST2, allowing the determination of soluble ST2 (sST2) in plasma samples from healthy individuals and patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) in only 45 min once the immunoconjugates have been prepared. The good correlation of the obtained results with those provided by an ELISA kit performed using the same immunoreagents demonstrates the potential of the developed strategy for early diagnosis and/or prognosis of the fatal PDAC disease.

Electrochemical immunoplatform to assist in the diagnosis and classification of breast cancer through the determination of matrix-metalloproteinase-9.

Matrix metalloproteinase 9 (MMP-9) is a zinc-dependent endopeptidase that promotes angiogenesis, tumor growth, metastasis and cell invasion through the degradation of extracellular matrix. This work reports a magnetic microbeads (MBs)-based sandwich immunoassay for the amperometric determination of MMP-9 at screen-printed carbon electrodes (SPCEs). The suitable capture antibody (cAb) is immobilized onto carboxylic MBs to selectively capture the antigen which is sandwiched with a biotinylated detector antibody (biotin-dAb) further conjugated with a commercial streptavidin-horseradish peroxidase (Strep-HRP) polymer. This immunoplatform provides great analytical characteristics in terms of selectivity and sensitivity, achieving a LOD value of 2.4 pg mL-1 for standards in buffered solutions. Although this value is similar to those reported for some other approaches described so far, the method described here is simpler involving a single 30 min incubation step which makes it ideal for automation or implementation in POC devices. Moreover, the method was assayed for the accurate determination of endogenous MMP-9 in both cancer cell lysates and serum samples of patients diagnosed with different subtypes of breast cancer (BC) after a simple dilution. The results obtained show that the disposable and affordable immunoplatform developed is able not only to discriminate BC patients from healthy individuals but also to do it for the worst outcome triple negative (TNBC) subtype.

Magnetic microbeads-based amperometric immunoplatform for the rapid and sensitive detection of N6-methyladenosine to assist in metastatic cancer cells discrimination.

This work describes the preparation of an immunoplatform for the sensitive and selective determination of N6-methyladenosine (m6A). The simple and fast protocol involves for the first time the use of micromagnetic immunoconjugates to establish a direct competitive assay between the m6A target and a biotinylated RNA oligomer bearing a single m6A enzymatically labelled with a commercial conjugate of streptavidin-peroxidase (Strep-HRP) as tracer. The cathodic current change measured in the presence of H2O2/hydroquinone (HQ) at screen-printed carbon electrodes (SPCEs) upon surface capturing the magnetic bioconjugates is inversely proportional to the m6A target concentration. After evaluating the effect of key variables, the analytical characteristics were established for the determination of three different targets: the N6-methyladenosine-5'-triphosphate (m6ATP) ribonucleotide, a short synthetic RNA oligomer bearing a single m6A and the positive control provided in a commercial colorimetric kit for m6A-RNA quantification. The obtained results show that this immunoplatform is competitive with other methods reported to date, achieving an improved sensitivity (limit of detection of 0.9 pM for the short synthetic oligomer) using a much simpler and faster protocol (~1 h) and disposable electrodes for the transduction. Furthermore, the applicability for discriminating the metastatic potential of cancer cells by directly analyzing a small amount of raw total RNA without enriching or fragmenting was also preliminary assessed.

TGF-ß-induced IGFBP-3 is a key paracrine factor from activated pericytes that promotes colorectal cancer cell migration and invasion.

The crosstalk between cancer cells and the tumor microenvironment has been implicated in cancer progression and metastasis. Fibroblasts and immune cells are widely known to be attracted to and modified by cancer cells. However, the role of pericytes in the tumor microenvironment beyond endothelium stabilization is poorly understood. Here, we report that pericytes promoted colorectal cancer (CRC) cell proliferation, migration, invasion, stemness, and chemoresistance in vitro, as well as tumor growth in a xenograft CRC model. We demonstrate that coculture with human CRC cells induced broad transcriptomic changes in pericytes, mostly associated with TGF-ß receptor activation. The prognostic value of a TGF-ß response signature in pericytes was analyzed in CRC patient data sets. This signature was found to be a good predictor of CRC relapse. Moreover, in response to stimulation by CRC cells, pericytes expressed high levels of TGF-ß1, initiating an autocrine activation loop. Investigation of secreted mediators and underlying molecular mechanisms revealed that IGFBP-3 is a key paracrine factor from activated pericytes affecting CRC cell migration and invasion. In summary, we demonstrate that the interplay between pericytes and CRC cells triggers a vicious cycle that stimulates pericyte cytokine secretion, in turn increasing CRC cell tumorigenic properties. Overall, we provide another example of how cancer cells can manipulate the tumor microenvironment.

Determination of miRNAs in serum of cancer patients with a label- and enzyme-free voltammetric biosensor in a single 30-min step.

The preparation of an integrated biosensor for the easy, fast, and sensitive determination of miRNAs is described based on a direct hybridization format and a label-free voltammetric detection. The biosensor involves a disposable carbon electrode substrate doubly nanostructured with reduced graphene oxide (rGO) and AuNPs modified with pyrene carboxylic acid (PCA) and 6-ferrocenylhexanethiol (Fc-SH), respectively. A synthetic amino terminated DNA capture probe was covalently immobilized on the CO2H moieties of PCA/rGO, while Fc-SH was used as a signaling molecule. Differential pulse voltammetry was employed to record the decrease in the oxidation peak current of Fc after the hybridization due to the hindering of the electron transfer upon the formation of the DNA-RNA duplex on the electrode surface. The stepwise biosensor preparation was characterized by surface and electrochemical techniques showing the role played by each biosensor component as well as the reliability of the target miRNA determination. The determination of the oncogene miRNA-21 synthetic target allowed quantification in the low femtomolar range (LOD of 5 fM) with a high discrimination of single-base mismatched sequences in a single 30-min incubation step. The bioplatform allowed the determination of the target miRNA in a small amount of total RNA extracted from breast cancer (BC) cells or directly in serum samples collected from BC patients without the need for prior extraction, purification, amplification, or reverse transcription of the genetic material and with no matrix effect. Graphical abstract.

Multiplexed monitoring of a novel autoantibody diagnostic signature of colorectal cancer using HaloTag technology-based electrochemical immunosensing platform.

Background and Purpose: The humoral immune response in cancer patients can be used for early detection of the disease. Autoantibodies raised against tumor-associated antigens (TAAs) are promising clinical biomarkers for reliable cancer diagnosis, prognosis, and therapy monitoring. In this study, an electrochemical disposable multiplexed immunosensing platform able to integrate difficult- and easy-to-express colorectal cancer (CRC) TAAs is reported for the sensitive determination of eight CRC-specific autoantibodies. Methods: The electrochemical immunosensing approach involves the use of magnetic microcarriers (MBs) as solid supports modified with covalently immobilized HaloTag fusion proteins for the selective capture of specific autoantibodies. After magnetic capture of the modified MBs onto screen-printed carbon working electrodes, the amperometric responses measured using the hydroquinone (HQ)/H2O2 system were related to the levels of autoantibodies in plasma. Results: The biosensing platform was applied to the analysis of autoantibodies against 8 TAAs described for the first time in this work in plasma samples from healthy asymptomatic individuals (n=3), and patients with high-risk of developing CRC (n=3), and from patients already diagnosed with colorectal (n=3), lung (n=2) or breast (n=2) cancer. The developed bioplatform demonstrated an improved discrimination between CRC patients and controls (asymptomatic healthy individuals and breast and lung cancer patients) compared to an ELISA-like luminescence test. Conclusions: The proposed methodology uses a just-in-time produced protein in a simpler protocol, with low sample volume, and involves cost-effective instrumentation, which could be used in a high-throughput manner for reliable population screening to facilitate the detection of early CRC patients at affordable cost.

Amperometric Bioplatforms To Detect Regional DNA Methylation with Single-Base Sensitivity.

This work reports the first bioplatform able to determine electrochemically 5-hydroxymethylcytosine (5-hmC) methylation events at localized sites and single-base sensitivity. The described bioplatform relies on a specific antibody (anti-5-hmC), further conjugated with commercial bioreagents loaded with multiple horseradish peroxidase (HRP) molecules, recognizing the epimark in a target DNA, captured through hybridization onto streptavidin-magnetic microbeads (Strep-MBs) modified with a complementary DNA capture probe. The electrochemical detection is performed by amperometry (-0.20 V vs Ag pseudoreference electrode) at disposable screen-printed carbon electrodes (SPCEs) in the presence of H2O2/hydroquinone (HQ) upon magnetic capture of the modified MBs onto the SPCE. The use of the commercial bioreagents ProtA-polyHRP80 and Histostar, very scarcely explored so far in electrochemical biosensors, provides high sensitivities for a synthetic target DNA sequence with a unique 5-hmC in the promoter region of MGMT tumor suppressor gene. Amplification factors of 43.6 and 55.2 were achieved using ProtA-polyHRP80 or Histostar, respectively, compared to the conventional secondary antibody labeling. This amplification was crucial to detect methylation events at single-nucleotide resolution achieving limits of detection (LODs) of 23.0 and 13.2 pM, respectively, without any target DNA amplification. The ProtA-polyHRP80-based bioplatform, selected as a compromise between sensitivity and cost per determination, exhibited full discrimination toward the target 5-hmC against the closely related 5-mC. In addition, the bioplatform detected 5-hmC at the regional level (MGMT promoter region) in just 10 ng of genomic DNA (gDNA, ∼2700 genomes) extracted from cancer cells and tissues from colorectal cancer (CRC) patients within 60 min.