RESUMO
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.
Assuntos
Técnicas Biossensoriais , Neoplasias Colorretais , MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/análise , Epigenoma , Hibridização de Ácido Nucleico/métodos , Anticorpos/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Prognóstico , Técnicas Biossensoriais/métodosRESUMO
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.
Assuntos
Ácidos Nucleicos , Adenosina , Humanos , Fenômenos Magnéticos , Metilação , RNARESUMO
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.
Assuntos
Técnicas Biossensoriais , Neoplasias , Adenosina/análogos & derivados , Peróxido de Hidrogênio , Limite de Detecção , Fenômenos Magnéticos , MicroesferasRESUMO
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.
Assuntos
Anticorpos Antineoplásicos/sangue , Autoanticorpos/sangue , Técnicas Biossensoriais , Neoplasias Colorretais/diagnóstico , Técnicas Eletroquímicas/métodos , Especificidade de Anticorpos , Antígenos de Neoplasias/imunologia , Área Sob a Curva , Doenças Assintomáticas , Biomarcadores Tumorais , Neoplasias da Mama/sangue , Neoplasias Colorretais/sangue , Neoplasias Colorretais/imunologia , Técnicas Eletroquímicas/instrumentação , Eletrodos , Feminino , Humanos , Hidroquinonas , Proteínas Imobilizadas/imunologia , Neoplasias Pulmonares/sangue , Masculino , Curva ROC , Proteínas Recombinantes de Fusão/imunologia , Sensibilidade e EspecificidadeRESUMO
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, â¼2700 genomes) extracted from cancer cells and tissues from colorectal cancer (CRC) patients within 60 min.
Assuntos
Metilação de DNA , Eletroquímica/métodos , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Linhagem Celular Tumoral , Humanos , Limite de DetecçãoRESUMO
This paper reports a simple electrochemical strategy for the determination of microRNAs (miRNAs) using a commercial His-Tag-Zinc finger protein (His-Tag-ZFP) that binds preferably (but non-sequence specifically) RNA hybrids over ssRNAs, ssDNAs, and dsDNAs. The strategy involves the use of magnetic beads (His-Tag-Isolation-MBs) as solid support to capture the conjugate formed in homogenous solution between His-Tag-ZFP and the dsRNA homohybrid formed between the target miRNA (miR-21 selected as a model) and a biotinylated synthetic complementary RNA detector probe (b-RNA-Dp) further conjugated with a streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The electrochemical detection is carried out by amperometry at disposable screen-printed carbon electrodes (SPCEs) (- 0.20 V vs Ag pseudo-reference electrode) upon magnetic capture of the resultant magnetic bioconjugates and H2O2 addition in the presence of hydroquinone (HQ). The as-prepared biosensor exhibits a dynamic concentration range from 3.0 to 100 nM and a detection limit (LOD) of 0.91 nM for miR-21 in just ~ 2 h. An acceptable discrimination was achieved between the target miRNA and other non-target nucleic acids (ssDNA, dsDNA, ssRNA, DNA-RNA, miR-122, miR-205, and single central- or terminal-base mismatched sequences). The biosensor was applied to the analysis of miR-21 from total RNA (RNAt) extracted from epithelial non-tumorigenic and adenocarcinoma breast cells without target amplification, pre-concentration, or reverse transcription steps. The versatility of the methodology due to the ZFP's non-sequence-specific binding behavior makes it easily extendable to determine any target RNA only by modifying the biotinylated detector probe.
Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , MicroRNAs/análise , Dedos de Zinco , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Limite de DetecçãoRESUMO
This paper reports the preparation of versatile electrochemical biosensing platforms for the simple, rapid, and PCR-independent detection of the most frequent DNA methylation marks (5-methylcytosine, 5-mC, and/or 5-hydroxymethylcytosine, 5-hmC) both at global and gene-specific levels. The implemented strategies, relying on the smart coupling of immuno-magnetic beads (MBs), specific DNA probes and amperometric detection at screen-printed carbon electrodes (SPCEs), provided sensitive and selective determination of the target methylated DNAs in less than 90 min with a great reproducibility and demonstrated feasibility for the simultaneous detection of the same or different cytosine epimarks both at global level and in different loci of the same gene or in different genes. The bioplatforms were applied to determine global methylation events in paraffin-embedded colorectal tissues and specific methylation at promoters of tumor suppressor genes in genomic DNA extracted from cancer cells and paraffin-embedded colorectal tissues, and in serum without previous DNA extraction from cancer patients.
Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/sangue , Biomarcadores Tumorais/sangue , Metilação de DNA , DNA/sangue , 5-Metilcitosina/imunologia , Anticorpos Monoclonais/imunologia , Armoracia/enzimologia , Biomarcadores Tumorais/química , Biomarcadores Tumorais/imunologia , Técnicas Biossensoriais/métodos , DNA/química , DNA/imunologia , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Técnicas Eletroquímicas/métodos , Corantes Fluorescentes/química , Peroxidase do Rábano Silvestre/química , Humanos , Peróxido de Hidrogênio/química , Separação Imunomagnética , Limite de Detecção , Proteínas Supressoras de Tumor/genéticaRESUMO
This paper reports the development of an amperometric immunosensing platform for the determination of cadherin-17 (CDH-17), an atypical adhesion protein involved in the progression, metastatic potential, and survival of high prevalence gastric, hepatocellular, and colorectal tumors. The methodology developed relies on the efficient capture and enzymatic labeling of the target protein on the magnetic microparticles (MBs) surface using commercial antibodies and amperometric transduction at screen-printed carbon electrodes (SCPEs) through the HRP/H2O2/HQ system. The developed immunosensing platform allows the selective determination of the target protein at low ng mL-1 level (LOD of 1.43 ng mL-1) in 45 min and using a single incubation step. The electrochemical immunosensor was successfully used for the accurate determination of the target protein in a small amount (0.5 µg) of raw lysates of colon cancer cells with different metastatic potential as well as in extracts from paraffin embedded cancer colon tissues of different metastatic grade.
Assuntos
Caderinas/análise , Neoplasias do Colo/patologia , Técnicas Eletroquímicas/métodos , Neoplasias Hepáticas/patologia , Neoplasias Gástricas/patologia , Técnicas Biossensoriais/métodos , Humanos , Peróxido de Hidrogênio/química , Hidroquinonas/química , Imunoensaio/métodos , Metástase Neoplásica/patologiaRESUMO
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 3.9-100â¯ngâ¯mL-1 concentration range, a LOD of 1.2â¯ngâ¯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.
Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Neoplasias do Colo/diagnóstico , Técnicas Eletroquímicas , Imunoensaio , Subunidade alfa2 de Receptor de Interleucina-13/análise , Neoplasias do Colo/patologia , Eletrodos , Humanos , Peróxido de Hidrogênio/química , Limite de Detecção , Reprodutibilidade dos TestesRESUMO
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 500â pm and a limit of detection of 1.2â 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.
Assuntos
5-Metilcitosina/análise , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Técnicas Eletroquímicas/métodos , Proteínas Supressoras de Tumor/genética , 5-Metilcitosina/sangue , 5-Metilcitosina/imunologia , Anticorpos/química , Anticorpos/imunologia , Técnicas Biossensoriais , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Eletrodos , Glioblastoma/diagnóstico , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Limite de Detecção , Hibridização de Ácido Nucleico , Regiões Promotoras GenéticasRESUMO
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.
Assuntos
Técnicas Biossensoriais , Metilação de DNA , Técnicas Eletroquímicas/instrumentação , Sulfatos/química , Líquidos Corporais/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Eletrodos , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Limite de Detecção , Proteínas Supressoras de Tumor/genéticaRESUMO
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.
Assuntos
Técnicas Biossensoriais , Eletrodos , Peroxidase do Rábano Silvestre , Humanos , Campos Magnéticos , MicroRNAs , Neoplasias , Hibridização de Ácido NucleicoRESUMO
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.
Assuntos
Biomarcadores Tumorais/análise , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , MicroRNAs/análise , Neoplasias/diagnóstico , Anticorpos Antinucleares/química , Carbono/química , DNA Complementar/química , Eletrodos , Humanos , Limite de Detecção , Células MCF-7 , MicroRNAs/química , Hibridização de Ácido Nucleico/métodos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
A novel nanocomposite material consisting of reduced graphene oxide/Rh nanoparticles was prepared by a one-pot reaction process. The strategy involved the simultaneous reduction of RhCl3 and graphene oxide with NaBH4 and the in situ deposition of the metal nanoparticles on the 2D carbon nanomaterial planar sheets. Glassy carbon electrode coated with this nanocomposite was employed as nanostructured support for the cross-linking of the enzyme laccase with glutaraldehyde to construct a voltammperometric biosensor for 17ß-estradiol in the 0.9-11 pM range. The biosensor showed excellent analytical performance with high sensitivity of 25.7AµM-1cm-1, a very low detection limit of 0.54pM and high selectivity. The biosensor was applied to the rapid and successful determination of the hormone in spiked synthetic and real human urine samples.