Gold Nanoparticle-Decorated Catalytic Micromotor-Based Aptassay for Rapid Electrochemical Label-Free Amyloid-ß42 Oligomer Determination in Clinical Samples from Alzheimer's Patients.

Micromotor (MM) technology offers a valuable and smart on-the-move biosensing microscale approach in clinical settings where sample availability is scarce in the case of Alzheimer's disease (AD). Soluble amyloid-ß protein oligomers (AßO) (mainly AßO42) that circulate in biological fluids have been recognized as a molecular biomarker and therapeutic target of AD due to their high toxicity, and they are correlated much more strongly with AD compared to the insoluble Aß monomers. A graphene oxide (GO)-gold nanoparticles (AuNPs)/nickel (Ni)/platinum nanoparticles (PtNPs) micromotors (MMGO-AuNPs)-based electrochemical label-free aptassay is proposed for sensitive, accurate, and rapid determination of AßO42 in complex clinical samples such as brain tissue, cerebrospinal fluid (CSF), and plasma from AD patients. An approach that implies the in situ formation of AuNPs on the GO external layer of tubular MM in only one step during MM electrosynthesis was performed (MMGO-AuNPs). The AßO42 specific thiolated-aptamer (AptAßO42) was immobilized in the MMGO-AuNPs via Au-S interaction, allowing for the selective recognition of the AßO42 (MMGO-AuNPs-AptAßO42-AßO42). AuNPs were smartly used not only to covalently bind a specific thiolated-aptamer for the design of a label-free electrochemical aptassay but also to improve the final MM propulsion performance due to their catalytic activity (approximately 2.0× speed). This on-the-move bioplatform provided a fast (5 min), selective, precise (RSD < 8%), and accurate quantification of AßO42 (recoveries 94-102%) with excellent sensitivity (LOD = 0.10 pg mL-1) and wide linear range (0.5-500 pg mL-1) in ultralow volumes of the clinical sample of AD patients (5 µL), without any dilution. Remarkably, our MM-based bioplatform demonstrated the competitiveness for the determination of AßO42 in the target samples against the dot blot analysis, which requires more than 14 h to provide qualitative results only. It is also important to highlight its applicability to the potential analysis of liquid biopsies as plasma and CSF samples, improving the reliability of the diagnosis given the heterogeneity and temporal complexity of neurodegenerative diseases. The excellent results obtained demonstrate the analytical potency of our approach as a future tool for clinical/POCT (Point-of-care testing) routine scenarios.

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.

Functional Proteomics Characterization of the Role of SPRYD7 in Colorectal Cancer Progression and Metastasis.

SPRY domain-containing protein 7 (SPRYD7) is a barely known protein identified via spatial proteomics as being upregulated in highly metastatic-to-liver KM12SM colorectal cancer (CRC) cells in comparison to its isogenic poorly metastatic KM12C CRC cells. Here, we aimed to analyze SPRYD7's role in CRC via functional proteomics. Through immunohistochemistry, the overexpression of SPRYD7 was observed to be associated with the poor survival of CRC patients and with an aggressive and metastatic phenotype. Stable SPRYD7 overexpression was performed in KM12C and SW480 poorly metastatic CRC cells and in their isogenic highly metastatic-to-liver-KM12SM-and-to-lymph-nodes SW620 CRC cells, respectively. Upon upregulation of SPRYD7, in vitro and in vivo functional assays confirmed a key role of SPRYD7 in the invasion and migration of CRC cells and in liver homing and tumor growth. Additionally, transient siRNA SPRYD7 silencing allowed us to confirm in vitro functional results. Furthermore, SPRYD7 was observed as an inductor of angiogenesis. In addition, the dysregulated SPRYD7-associated proteome and SPRYD7 interactors were elucidated via 10-plex TMT quantitative proteins, immunoproteomics, and bioinformatics. After WB validation, the biological pathways associated with the stable overexpression of SPRYD7 were visualized. In conclusion, it was demonstrated here that SPRYD7 is a novel protein associated with CRC progression and metastasis. Thus, SPRYD7 and its interactors might be of relevance in identifying novel therapeutic targets for advanced CRC.

Benefits of FAIMS to Improve the Proteome Coverage of Deteriorated and/or Cross-Linked TMT 10-Plex FFPE Tissue and Plasma-Derived Exosomes Samples.

The proteome characterization of complex, deteriorated, or cross-linked protein mixtures as paired clinical FFPE or exosome samples isolated from low plasma volumes (250 µL) might be a challenge. In this work, we aimed at investigating the benefits of FAIMS technology coupled to the Orbitrap Exploris 480 mass spectrometer for the TMT quantitative proteomics analyses of these complex samples in comparison to the analysis of protein extracts from cells, frozen tissue, and exosomes isolated from large volume plasma samples (3 mL). TMT experiments were performed using a two-hour gradient LC-MS/MS with or without FAIMS and two compensation voltages (CV = -45 and CV = -60). In the TMT experiments of cells, frozen tissue, or exosomes isolated from large plasma volumes (3 mL) with FAIMS, a limited increase in the number of identified and quantified proteins accompanied by a decrease in the number of peptides identified and quantified was observed. However, we demonstrated here a noticeable improvement (>100%) in the number of peptide and protein identifications and quantifications for the plasma exosomes isolated from low plasma volumes (250 µL) and FFPE tissue samples in TMT experiments with FAIMS in comparison to the LC-MS/MS analysis without FAIMS. Our results highlight the potential of mass spectrometry analyses with FAIMS to increase the depth into the proteome of complex samples derived from deteriorated, cross-linked samples and/or those where the material was scarce, such as FFPE and plasma-derived exosomes from low plasma volumes (250 µL), which might aid in the characterization of their proteome and proteoforms and in the identification of dysregulated proteins that could be used as biomarkers.

Brain and immune system-derived extracellular vesicles mediate regulation of complement system, extracellular matrix remodeling, brain repair and antigen tolerance in Multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated central nervous system disease whose course is unpredictable. Finding biomarkers that help to better comprehend the disease's pathogenesis is crucial for supporting clinical decision-making. Blood extracellular vesicles (EVs) are membrane-bound particles secreted by all cell types that contain information on the disease's pathological processes. PURPOSE: To identify the immune and nervous system-derived EV profile from blood that could have a specific role as biomarker in MS and assess its possible correlation with disease state. RESULTS: Higher levels of T cell-derived EVs and smaller size of neuron-derived EVs were associated with clinical relapse. The smaller size of the oligodendrocyte-derived EVs was related with motor and cognitive impairment. The proteomic analysis identified mannose-binding lectin serine protease 1 and complement factor H from immune system cell-derived EVs as autoimmune disease-associated proteins. We observed hepatocyte growth factor-like protein in EVs from T cells and inter-alpha-trypsin inhibitor heavy chain 2 from neurons as white matter injury-related proteins. In patients with MS, a specific protein profile was found in the EVs, higher levels of alpha-1-microglobulin and fibrinogen ß chain, lower levels of C1S and gelsolin in the immune system-released vesicles, and Talin-1 overexpression in oligodendrocyte EVs. These specific MS-associated proteins, as well as myelin basic protein in oligodendrocyte EVs, correlated with disease activity in the patients with MS. CONCLUSION: Neural-derived and immune-derived EVs found in blood appear to be good specific biomarkers in MS for reflecting the disease state.

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.

Proteomics analysis of prefrontal cortex of Alzheimer's disease patients revealed dysregulated proteins in the disease and novel proteins associated with amyloid-ß pathology.

BACKGROUND: Alzheimer's disease (AD) is a progressive, chronic, and neurodegenerative disease, and the most common cause of dementia worldwide. Currently, the mechanisms underlying the disease are far from being elucidated. Thus, the study of proteins involved in its pathogenesis would allow getting further insights into the disease and identifying new markers for AD diagnosis. METHODS: We aimed here to analyze protein dysregulation in AD brain by quantitative proteomics to identify novel proteins associated with the disease. 10-plex TMT (tandem mass tags)-based quantitative proteomics experiments were performed using frozen tissue samples from the left prefrontal cortex of AD patients and healthy individuals and vascular dementia (VD) and frontotemporal dementia (FTD) patients as controls (CT). LC-MS/MS analyses were performed using a Q Exactive mass spectrometer. RESULTS: In total, 3281 proteins were identified and quantified using MaxQuant. Among them, after statistical analysis with Perseus (p value < 0.05), 16 and 155 proteins were defined as upregulated and downregulated, respectively, in AD compared to CT (Healthy, FTD and VD) with an expression ratio ≥ 1.5 (upregulated) or ≤ 0.67 (downregulated). After bioinformatics analysis, ten dysregulated proteins were selected as more prone to be associated with AD, and their dysregulation in the disease was verified by qPCR, WB, immunohistochemistry (IHC), immunofluorescence (IF), pull-down, and/or ELISA, using tissue and plasma samples of AD patients, patients with other dementias, and healthy individuals. CONCLUSIONS: We identified and validated novel AD-associated proteins in brain tissue that should be of further interest for the study of the disease. Remarkably, PMP2 and SCRN3 were found to bind to amyloid-ß (Aß) fibers in vitro, and PMP2 to associate with Aß plaques by IF, whereas HECTD1 and SLC12A5 were identified as new potential blood-based biomarkers of the disease.

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.

PKD phosphorylation and COP9/Signalosome modulate intracellular Spry2 protein stability.

Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.

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.

In-depth quantitative proteomics analysis revealed C1GALT1 depletion in ECC-1 cells mimics an aggressive endometrial cancer phenotype observed in cancer patients with low C1GALT1 expression.

BACKGROUND: Endometrial cancer (EC) is the most common cancer of the female reproductive organs. Despite the good overall prognosis of most low-grade ECs, FIGO I and FIGO II patients might experience tumor recurrence and worse prognosis. The study of alterations related to EC pathogenesis might help to get insights into underlying mechanisms involved in EC development and progression. METHODS: Core tumoral samples were used to investigate the role of C1GALT1 in EC by immunohistochemistry (IHC). ECC-1 cells were used as endometrioid EC model to investigate the effect of C1GALT1 depletion using C1GALT1 specific shRNAs. SILAC quantitative proteomics analyses and cell-based assays, PCR, qPCR, WB, dot-blot and IHC analyses were used to identify, quantify and validate dysregulation of proteins. RESULTS: Low C1GALT1 protein expression levels associate to a more aggressive phenotype of EC. Out of 5208 proteins identified and quantified by LC-MS/MS, 100 proteins showed dysregulation (log2fold-change ≥ 0.58 or ≤-0.58) in the cell protein extracts and 144 in the secretome of C1GALT1 depleted ECC-1 cells. Nine dysregulated proteins were validated. Bioinformatics analyses pointed out to an increase in pathways associated with an aggressive phenotype. This finding was corroborated by loss-of-function cell-based assays demonstrating higher proliferation, invasion, migration, colony formation and angiogenesis capacity in C1GALT1 depleted cells. These effects were associated to the overexpression of ANXA1, as demonstrated by ANXA1 transient silencing cell-based assays, and thus, correlating C1GALT and ANXA1 protein expression and biological effects. Finally, the negative protein expression correlation found by proteomics between C1GALT1 and LGALS3 was confirmed by IHC. CONCLUSION: C1GALT1 stably depleted ECC-1 cells mimic an EC aggressive phenotype observed in patients and might be useful for the identification and validation of EC markers of progression.

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.

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.

Metabolic Reprogramming Helps to Define Different Metastatic Tropisms in Colorectal Cancer.

Approximately 25% of colorectal cancer (CRC) patients experience systemic metastases, with the most frequent target organs being the liver and lung. Metabolic reprogramming has been recognized as one of the hallmarks of cancer. Here, metabolic and functional differences between two CRC cells with different metastatic organotropisms (metastatic KM12SM CRC cells to the liver and KM12L4a to the lung when injected in the spleen and in the tail vein of mice) were analysed in comparison to their parental non-metastatic isogenic KM12C cells, for a subsequent investigation of identified metabolic targets in CRC patients. Meta-analysis from proteomic and transcriptomic data deposited in databases, qPCR, WB, in vitro cell-based assays, and in vivo experiments were used to survey for metabolic alterations contributing to their different organotropism and for the subsequent analysis of identified metabolic markers in CRC patients. Although no changes in cell proliferation were observed between metastatic cells, KM12SM cells were highly dependent on oxidative phosphorylation at mitochondria, whereas KM12L4a cells were characterized by being more energetically efficient with lower basal respiration levels and a better redox management. Lipid metabolism-related targets were found altered in both cell lines, including LDLR, CD36, FABP4, SCD, AGPAT1, and FASN, which were also associated with the prognosis of CRC patients. Moreover, CD36 association with lung metastatic tropism of CRC cells was validated in vivo. Altogether, our results suggest that LDLR, CD36, FABP4, SCD, FASN, LPL, and APOA1 metabolic targets are associated with CRC metastatic tropism to the liver or lung. These features exemplify specific metabolic adaptations for invasive cancer cells which stem at the primary tumour.

Assisting dementia diagnosis through the electrochemical immunosensing of glial fibrillary acidic protein.

Glial fibrillary acidic protein (GFAP) is a member of the intermediate filament family of proteins with increased levels in serum and cerebrospinal fluid of patients with Alzheimer disease (AD) and other neurodegenerative diseases (NDs), such as vascular dementia (VD). This work describes the first magnetic microbeads (MBs)-based electrochemical immunoplatform for GFAP determination. The platform design comprises a sandwich immunoassay implemented on the MBs surface and amperometric transduction at single-use screen-printed carbon electrodes (SPCEs). Micro-sized carboxylic acid magnetic particles (COOH-MBs) were modified with a specific capture antibody (CAb) to selectively link the target protein, which was sandwiched with a biotinylated detector antibody (btn-DAb) further conjugated with a streptavidin-peroxidase (Strep-HRP) conjugate. Amperometric transduction was performed at SPCEs upon capturing the magnetic bioconjugates on their surface and through the hydrogen peroxide/hydroquinone (H2O2/HQ) system. The immunoplatform achieved a limit of detection of 67 pg mL-1 for the amperometric detection of standards and selectivity compatible with clinical applicability to assist in minimally invasive NDs diagnosis and prognosis. The MBs-based immunoplatform was applied with good results to determine the endogenous content of GFAP in protein brain extracts without matrix effect and using just 6.25 ng of sample per determination. Furthermore, the developed methodology was capable of differentiating between healthy subjects and patients diagnosed with VD and AD in only 2 h, providing accurate results in line with those obtained by an ELISA kit that used the same immunoreagents.

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.

In-depth proteomics characterization of ∆Np73 effectors identifies key proteins with diagnostic potential implicated in lymphangiogenesis, vasculogenesis and metastasis in colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death worldwide. Alterations in proteins of the p53-family are a common event in CRC. ΔNp73, a p53-family member, shows oncogenic properties and its effectors are largely unknown. We performed an in-depth proteomics characterization of transcriptional control by ∆Np73 of the secretome of human colon cancer cells and validated its clinical potential. The secretome was analyzed using high-density antibody microarrays and stable isotopic metabolic labeling. Validation was performed by semiquantitative PCR, ELISA, dot-blot and western blot analysis. Evaluation of selected effectors was carried out using 60 plasma samples from CRC patients, individuals carrying premalignant colorectal lesions and colonoscopy-negative controls. In total, 51 dysregulated proteins were observed showing at least 1.5-foldchange in expression. We found an important association between the overexpression of ∆Np73 and effectors related to lymphangiogenesis, vasculogenesis and metastasis, such as brain-derived neurotrophic factor (BDNF) and the putative aminoacyl tRNA synthase complex-interacting multifunctional protein 1 (EMAP-II)-vascular endothelial growth factor C-vascular endothelial growth factor receptor 3 axis. We further demonstrated the usefulness of BDNF as a potential CRC biomarker able to discriminate between CRC patients and premalignant individuals from controls with high sensitivity and specificity.

Aryl-hydrocarbon receptor-interacting protein regulates tumorigenic and metastatic properties of colorectal cancer cells driving liver metastasis.

BACKGROUND: Liver metastasis is the primary cause of colorectal cancer (CRC)-associated death. Aryl-hydrocarbon receptor-interacting protein (AIP), a putative positive intermediary in aryl-hydrocarbon receptor-mediated signalling, is overexpressed in highly metastatic human KM12SM CRC cells and other highly metastatic CRC cells. METHODS: Meta-analysis and immunohistochemistry were used to assess the relevance of AIP. Cellular functions and signalling mechanisms mediated by AIP were assessed by gain-of-function experiments and in vitro and in vivo experiments. RESULTS: A significant association of high AIP expression with poor CRC patients' survival was observed. Gain-of-function and quantitative proteomics experiments demonstrated that AIP increased tumorigenic and metastatic properties of isogenic KM12C (poorly metastatic) and KM12SM (highly metastatic to the liver) CRC cells. AIP overexpression dysregulated epithelial-to-mesenchymal (EMT) markers and induced several transcription factors and Cadherin-17 activation. The former induced the signalling activation of AKT, SRC and JNK kinases to increase adhesion, migration and invasion of CRC cells. In vivo, AIP expressing KM12 cells induced tumour growth and liver metastasis. Furthermore, KM12C (poorly metastatic) cells ectopically expressing AIP became metastatic to the liver. CONCLUSIONS: Our data reveal new roles for AIP in regulating proteins associated with cancer and metastasis to induce tumorigenic and metastatic properties in colon cancer cells driving liver metastasis.

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.