Víctor Ruiz-Valdepeñas Montiel

Toward Liquid Biopsy: Determination of the Humoral Immune Response in Cancer Patients Using HaloTag Fusion Protein-Modified Electrochemical Bioplatforms

Autoantibodies raised against tumor-associated antigens have shown high promise as clinical biomarkers for reliable diagnosis, prognosis, and therapy monitoring of cancer. An electrochemical disposable biosensor for the specific and sensitive determination of p53-specific autoantibodies has been developed for the first time in this work. This biosensor involves the use of magnetic microcarriers (MBs) modified with covalently immobilized HaloTag fusion p53 protein as solid supports for the selective capture of specific autoantibodies. After magnetic capture of the modified MBs onto screen-printed carbon working electrodes, the amperometric signal using the system hydroquinone/H2O2 was related to the levels of p53-autoantibodies in the sample. The biosensor was applied for the analysis of sera from 24 patients with high-risk of developing colorectal cancer and 6 from patients already diagnosed with colorectal (4) and ovarian (2) cancer. The developed biosensor was able to determine p53 autoantibodies with a sensitivity higher than that of a commercial standard ELISA using a just-in-time produced protein in a simpler protocol with less sample volume and easily miniaturized and cost-effective instrumentation.

Magnetic Beads-Based Sensor with Tailored Sensitivity for Rapid and Single-Step Amperometric Determination of miRNAs

This work describes a sensitive amperometric magneto-biosensor for single-step and rapid determination of microRNAs (miRNAs). The developed strategy involves the use of direct hybridization of the target miRNA (miRNA-21) with a specific biotinylated DNA probe immobilized on streptavidin-modified magnetic beads (MBs), and labeling of the resulting heteroduplexes with a specific DNA–RNA antibody and the bacterial protein A (ProtA) conjugated with an horseradish peroxidase (HRP) homopolymer (Poly-HRP40) as an enzymatic label for signal amplification. Amperometric detection is performed upon magnetic capture of the modified MBs onto the working electrode surface of disposable screen-printed carbon electrodes (SPCEs) using the H2O2/hydroquinone (HQ) system. The magnitude of the cathodic signal obtained at −0.20 V (vs. the Ag pseudo-reference electrode) demonstrated linear dependence with the concentration of the synthetic target miRNA over the 1.0 to 100 pM range. The method provided a detection limit (LOD) of 10 attomoles (in a 25 μL sample) without any target miRNA amplification in just 30 min (once the DNA capture probe-MBs were prepared). This approach shows improved sensitivity compared with that of biosensors constructed with the same anti-DNA–RNA Ab as capture instead of a detector antibody and further labeling with a Strep-HRP conjugate instead of the Poly-HRP40 homopolymer. The developed strategy involves a single step working protocol, as well as the possibility to tailor the sensitivity by enlarging the length of the DNA/miRNA heteroduplexes using additional probes and/or performing the labelling with ProtA conjugated with homopolymers prepared with different numbers of HRP molecules. The practical usefulness was demonstrated by determination of the endogenous levels of the mature target miRNA in 250 ng raw total RNA (RNAt) extracted from human mammary epithelial normal (MCF-10A) and cancer (MCF-7) cells and tumor tissues.

Electrochemical affinity biosensors for fast detection of gene-specific methylations with no need for bisulfite and amplification treatments

This paper describes two different electrochemical affinity biosensing approaches for the simple, fast and bisulfite and PCR-free quantification of 5-methylated cytosines (5-mC) in DNA using the anti-5-mC antibody as biorecognition element. One of the biosensing approaches used the anti-5-mC as capture bioreceptor and a sandwich type immunoassay, while the other one involved the use of a specific DNA probe and the anti-5-mC as a detector bioreceptor of the captured methylated DNA. Both strategies, named for simplicity in the text as immunosensor and DNA sensor, respectively, were implemented on the surface of magnetic microparticles and the transduction was accomplished by amperometry at screen-printed carbon electrodes by means of the hydrogen peroxide/hydroquinone system. The resulting amperometric biosensors demonstrated reproducibility throughout the entire protocol, sensitive determination with no need for using amplification strategies, and competitiveness with the conventional enzyme-linked immunosorbent assay methodology and the few electrochemical biosensors reported so far in terms of simplicity, sensitivity and assay time. The DNA sensor exhibited higher sensitivity and allowed the detection of the gene-specific methylations conversely to the immunosensor, which detected global DNA methylation. In addition, the DNA sensor demonstrated successful applicability for 1 h-analysis of specific methylation in two relevant tumor suppressor genes in spiked biological fluids and in genomic DNA extracted from human glioblastoma cells.

Comparison of Different Strategies for the Development of Highly Sensitive Electrochemical Nucleic Acid Biosensors Using Neither Nanomaterials nor Nucleic Acid Amplification

Currently, electrochemical nucleic acid-based biosensing methodologies involving hybridization assays, specific recognition of RNA/DNA and RNA/RNA duplexes, and amplification systems provide an attractive alternative to conventional quantification strategies for the routine determination of relevant nucleic acids at different settings. A particularly relevant objective in the development of such nucleic acid biosensors is the design of as many as possible affordable, quick, and simple methods while keeping the required sensitivity. With this aim in mind, this work reports, for the first time, a thorough comparison between 11 methodologies that involve different assay formats and labeling strategies for targeting the same DNA. The assayed approaches use conventional sandwich and competitive hybridization assays, direct hybridization coupled to bioreceptors with affinity for RNA/DNA duplexes, multienzyme labeling bioreagents, and DNA concatamers. All of them have been implemented on the surface of magnetic beads (MBs) and involve amperometric transduction at screen-printed carbon electrodes (SPCEs). The influence of the formed duplex length and of the labeling strategy have also been evaluated. Results demonstrate that these strategies can provide very sensitive methods without the need for using nanomaterials or polymerase chain reaction (PCR). In addition, the sensitivity can be tailored within several orders of magnitude simply by varying the bioassay format, hybrid length or labeling strategy. This comparative study allowed us to conclude that the use of strategies involving longer hybrids, the use of antibodies with specificity for RNA/DNA heteroduplexes and labeling with bacterial antibody binding proteins conjugated with multiple enzyme molecules, provides the best sensitivity.

Electrochemical sensor for rapid determination of fibroblast growth factor receptor 4 in raw cancer cell lysates

The first electrochemical immunosensor for the determination of fibroblast growth factor receptor 4 (FGFR4) biomarker is reported in this work. The biosensor involves a sandwich configuration with covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic microcarriers (HOOC-MBs) and amperometric detection at disposable carbon screen-printed electrodes (SPCEs). The biosensor exhibits a great analytical performance regarding selectivity for the target protein and a low LOD of 48.2 pg mL-1. The electrochemical platform was successfully applied for the determination of FGFR4 in different cancer cell lysates without any apparent matrix effect after a simple sample dilution and using only 2.5 μg of the raw lysate. Comparison of the results with those provided by a commercial ELISA kit shows competitive advantages by using the developed immunosensor in terms of simplicity, analysis time, and portability and cost-affordability of the required instrumentation for the accurate determination of FGFR4 in cell lysates.

Disposable Amperometric Polymerase Chain Reaction-Free Biosensor for Direct Detection of Adulteration with Horsemeat in Raw Lysates Targeting Mitochondrial DNA

A novel electrochemical disposable nucleic acid biosensor for simple, rapid, and specific detection of adulterations with horsemeat is reported in this work. The biosensing platform involves immobilization of a 40-mer RNA probe specific for a characteristic fragment of the mitochondrial DNA D-loop region of horse onto the surface of magnetic microcarriers. In addition, signal amplification was accomplished by using a commercial antibody specific to RNA/DNA duplexes and a bacterial protein conjugated with a horseradish peroxidase homopolymer (ProtA-HRP40). Amperometric detection at -0.20 V vs Ag pseudoreference electrode was carried out at disposable screen-printed carbon electrodes. The methodology achieved a limit of detection (LOD) of 0.12 pM (3.0 attomoles) for the synthetic target and showed ability to discriminate between raw beef and horsemeat using just 50 ng of total extracted mitochondrial DNA (∼16 660 bp in length) without previous fragmentation. The biosensor also allowed discrimination between 100% raw beef and beef meat samples spiked with only 0.5% (w/w) horse meat (levels established by the European Commission) using raw mitochondrial lysates without DNA extraction or polymerase chain reaction (PCR) amplification in just 75 min. These interesting features made the developed methodology an extremely interesting tool for beef meat screening, and it can be easily adapted to the determination of other meat adulterations by selection of the appropriate specific fragments of the mitochondrial DNA region and capture probes.

Electrochemical Biosensors for Food Security: Allergens and Adulterants Detection

Food safety plays an important role in public health and thus to society as a whole. Food may become unsafe due to the presence of allergens and adulterants amongst others and hence the determination of these analytes has gained great relevance in quality control and safety of food. Electrochemical biosensor devices are emerging as one of the foremost relevant techniques for monitoring food allergens and adulterants due to their quick, specific, sensitive and reliable performances, ease of mass fabrication, economics and field applicability. These electrochemical biosensors, based on the use of existing or recently developed bioreceptors in combination with nanomaterials and surface fabrication techniques are able to offer attractive and efficient platforms which may become a viable option for the development of simpler, faster, cheaper and more robust and reliable analytical methods for the detection of allergens and adulterants in food. This Chapter describes recent analytical strategies developed so far using electrochemical biosensors for the determination of potentially hazardous adulterants and allergenic food residues to ensure food safety. The main progresses achieved to date are presented, highlighting general considerations and potential prospects for the future. The variety of electrochemical biosensors that have appeared in recent years shows that it is a booming research area with still many challenges but also great opportunities to develop sensitive, reliable, robust, and cost effective food allergens and adulterants biosensing methodologies.

Rapid Electrochemical Assessment of Tumor Suppressor Gene Methylations in Raw Human Serum and Tumor Cells and Tissues Using Immunomagnetic Beads and Selective DNA Hybridization

We report a rapid and sensitive electrochemical strategy for the detection of gene-specific 5-methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody for 5-methylcytosines (5-mC) are used for the capture of any 5-mC methylated single-stranded (ss)DNA sequence. A flanking region next to the 5-mCs of the captured methylated ssDNA is recognized by hybridization with a synthetic biotinylated DNA sequence. Amperometric transduction at disposable screen-printed carbon electrodes (SPCEs) is employed. The developed biosensor has a dynamic range from 3.9 to 500 pm and a limit of detection of 1.2 pm for the methylated synthetic sequence of the tumor suppressor gene O-6-methylguanine-DNA methyltransferase (MGMT) promoter region. The method is applied in the 45-min analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U-87 glioblastoma cells and paraffin-embedded brain tumor tissues without any amplification and pretreatment step.

Single-Step Incubation Determination of miRNAs in Cancer Cells Using an Amperometric Biosensor Based on Competitive Hybridization onto Magnetic Beads

This work reports an amperometric biosensor for the determination of miRNA-21, a relevant oncogene. The methodology involves a competitive DNA-target miRNA hybridization assay performed on the surface of magnetic microbeads (MBs) and amperometric transduction at screen-printed carbon electrodes (SPCEs). The target miRNA competes with a synthetic fluorescein isothiocyanate (FITC)-modified miRNA with an identical sequence for hybridization with a biotinylated and complementary DNA probe (b-Cp) immobilized on the surface of streptavidin-modified MBs (b-Cp-MBs). Upon labeling, the FITC-modified miRNA attached to the MBs with horseradish peroxidase (HRP)-conjugated anti-FITC Fab fragments and magnetic capturing of the MBs onto the working electrode surface of SPCEs. The cathodic current measured at −0.20 V (versus the Ag pseudo-reference electrode) was demonstrated to be inversely proportional to the concentration of the target miRNA. This convenient biosensing method provided a linear range between 0.7 and 10.0 nM and a limit of detection (LOD) of 0.2 nM (5 fmol in 25 μL of sample) for the synthetic target miRNA without any amplification step. An acceptable selectivity towards single-base mismatched oligonucleotides, a high storage stability of the b-Cp-MBs, and usefulness for the accurate determination of miRNA-21 in raw total RNA (RNAt) extracted from breast cancer cells (MCF-7) were demonstrated.

Electrochemical immunosensor for IL-13 Receptor α2 determination and discrimination of metastatic colon cancer cells

This work describes the first electrochemical immunosensor reported for the determination of IL-13 receptor Rα2 (IL-13Rα2), an emerging relevant biomarker in metastatic colon cancer. The approach involves the formation of sandwich immunocomplexes using specific capture (CAb) and biotinylated detector antibodies (BDAb) further labeled with an streptavidin-horseradish peroxidase (Strep-HRP) polymer, onto carboxylic acid-modified magnetic microbeads (HOOC-MBs). Amperometric detection at disposable carbon screen-printed electrodes (SPCEs) using the (H2O2)/hydroquinone (HQ) system was employed to monitor the affinity reactions. The developed immunosensor exhibits a linear calibration plot over the 3.9-100 ng mL-1 concentration range, a LOD of 1.2 ng mL-1 and excellent selectivity against other non-target proteins. The amperometric immunosensor was applied successfully to quantify for the first time the IL-13Rα2 expression in raw lysates of colon cancer cells and to discriminate the metastatic potential of intact cells through recognition of this target extracellular receptor. In comparison with the commercial Enzyme-Linked ImmunoSorbent Assay (ELISA) kit involving the same immunoreagents, the immunosensor provides a similar LOD in a half-time for the assay.