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DNA-based enzymes, or DNAzymes, are single-stranded DNA sequences with the ability to catalyze various chemical reactions, including the cleavage of the bond between two RNA nucleotides. Lately, an increasing interest has been observed in these RNA-cleaving DNAzymes in the biosensing and therapeutic fields for signal generation and the modulation of gene expression, respectively. Additionally, multiple efforts have been made to study the effects of the reaction environment and the sequence of the catalytic core on the conversion of the substrate into product. However, most of these studies have only reported alterations of the general reaction course, but only a few have focused on how each individual reaction step is affected. In this work, we present for the first time a mathematical model that describes and predicts the reaction of the 10-23 RNA-cleaving DNAzyme. Furthermore, the model has been employed to study the effect of temperature, magnesium cations and shorter substrate-binding arms of the DNAzyme on the different kinetic rate constants, broadening the range of conditions in which the model can be exploited. In conclusion, this work depicts the prospects of such mathematical models to study and anticipate the course of a reaction given a particular environment.
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ADN Catalítico , Catálisis , Dominio Catalítico , ADN de Cadena Simple/genética , ARN/genéticaRESUMEN
Extracellular vesicles (EVs) have attracted great attention as potential biomarkers for cancer diagnostics. Although several technologies have been developed for EV detection, many of them are still not applicable to clinical settings as they rely on complex EV isolation processes, while lacking sensitivity, specificity or standardization. To solve this problem, we have developed a sensitive breast cancer-specific EV detection bioassay directly in blood plasma using a fiber-optic surface plasmon resonance (FO-SPR) biosensor, previously calibrated with recombinant EVs. First, we established a sandwich bioassay to detect SK-BR-3 EVs by functionalizing the FO-SPR probes with anti-HER2 antibodies. A calibration curve was built using an anti-HER2/Banti-CD9 combination, resulting in an LOD of 2.1 × 107 particles/mL in buffer and 7 × 108 particles/mL in blood plasma. Next, we investigated the potential of the bioassay to detect MCF7 EVs in blood plasma using an anti-EpCAM/Banti-mix combination, obtaining an LOD of 1.1 × 10 8 particles/mL. Finally, the specificity of the bioassay was proven by the absence of signal when testing plasma samples from 10 healthy people unknown to be diagnosed with breast cancer. The remarkable sensitivity and specificity of the developed sandwich bioassay together with the advantages of the standardized FO-SPR biosensor highlight outstanding potential for the future of EV analysis.
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Técnicas Biosensibles , Neoplasias de la Mama , Vesículas Extracelulares , Femenino , Humanos , Biomarcadores , Técnicas Biosensibles/métodos , Neoplasias de la Mama/diagnóstico , Resonancia por Plasmón de Superficie/métodosRESUMEN
Single cell analyses have gained increasing interest over bulk approaches because of considerable cell-to-cell variability within isogenic populations. Herein, flow cytometry remains golden standard due to its high-throughput efficiency and versatility, although it does not allow to investigate the interdependency of cellular events over time. Starting from our microfluidic platform that enables to trap and retain individual cells on a fixed location over time, here, we focused on unraveling kinetic responses of single Saccharomyces cerevisiae yeast cells upon treatment with the antifungal plant defensin HsAFP1. We monitored the time between production of reactive oxygen species (ROS) and membrane permeabilization (MP) in single yeast cells for different HsAFP1 doses using two fluorescent dyes with non-overlapping spectra. Within a time frame of 2 min, only <0.3% cells displayed time between the induction of ROS and MP. Reducing the time frame to 30 s did not result in increased numbers of cells with time between these events, pointing to ROS and MP induction as highly dynamic and correlated processes. In conclusion, using an in-house developed continuous microfluidic platform, we investigated the mode of action of HsAFP1 at single cell level, thereby uncovering the close interdependency between ROS induction and MP in yeast.
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Defensinas/farmacología , Fungicidas Industriales/farmacología , Heuchera/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Blanqueamiento de los Corales , Viabilidad Microbiana/efectos de los fármacos , Técnicas Analíticas Microfluídicas , Proteínas de Plantas/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Análisis de la Célula Individual , Factores de TiempoRESUMEN
Testing multiple biomarkers, as opposed to one, has become a preferred approach for diagnosing many heterogeneous diseases, such as cancer and infectious diseases. However, numerous technologies, including gold standard ELISA and PCR, can detect only one type of biomarker, either protein or nucleic acid (NA), respectively. In this work, we report for the first time simultaneous detection of proteins and NAs in the same solution, using solely functional NA (FNA) molecules. In particular, we combined the thrombin binding aptamer (TBA) and the 10-23 RNA-cleaving DNA enzyme (DNAzyme) in a single aptazyme molecule (Aptazyme1.15-3'), followed by extensive optimization of buffer composition, sequences and component ratios, to establish a competitive bioassay. Subsequently, to establish a multiplex bioassay, we designed a new aptazyme (Aptazyme2.20-5') by replacing the target recognition and substrate sequences within Aptazyme1.15-3'. This designing process included an in silico study, revealing the impact of the target recognition sequence on the aptazyme secondary structure and its catalytic activity. After proving the functionality of the new aptazyme in a singleplex bioassay, we demonstrated the capability of the two aptazymes to simultaneously detect thrombin and NA target, or two NA targets in a multiplex bioassay. High specificity in target detection was achieved with the limits of detection in the low nanomolar range, comparable to the singleplex bioassays. The presented results deepen the barely explored features of FNA for diagnosing multiple targets of different origins, adding an extra functionality to their catalogue.
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Bioensayo/métodos , Técnicas Biosensibles/métodos , ADN Catalítico/metabolismo , ADN/química , Ácidos Nucleicos/química , Trombina/química , ADN Catalítico/química , Humanos , Nanotecnología , Reproducibilidad de los ResultadosRESUMEN
The polymerase chain reaction (PCR) has been the gold standard molecular analysis technique for decades and has seen quite some evolution in terms of reaction components, methodology, and readout mechanisms. Nucleic acid enzymes (NAzymes) have been used to further exploit the applications of PCR, but so far the work was limited to the colorimetric G-quadruplex or fluorescent substrate cleaving NAzymes. In this study, a solid-phase, fiber optic surface plasmon resonance (FO-SPR) technique is presented as an alternative readout for PCR utilizing NAzymes. First, the surface cleavage activity of DNAzyme-extended amplicons (DNAzyme-amps) is established, followed by optimization of the PCR conditions, which are required for compatibility with the FO-SPR system. Next, by integrating the complement of a 10-23 DNAzyme into the primer pair, PCR-amplified DNAzyme-amps were generated, tested, and validated on qPCR for the detection of the antimicrobial resistance gene MCR-2. Once validated, this primer concept was developed as a one-step assay, driven by PCR-amplified DNAzymes, for FO-SPR-based sensitive and specific detection. Using gold nanoparticle labeled RNA-DNA hybrid strands as substrate for the DNAzyme, PCR-amplified DNAzyme-amps generated in the presence of MCR-2 gene were monitored in real-time, which resulted in an experimental limit of detection of 4 × 105 copy numbers or 6.6 fM. In addition, the DNAzyme-based FO-PCR assay was able to discriminate between the MCR-1 and MCR-2 genes, to further prove the specificity of this assay. Henceforth, this DNAzyme-based fiber optic PCR assay provides a universally applicable, real-time system for the detection of virtually any target NA, in a specific and sensitive manner.
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ADN Catalítico/genética , ADN Catalítico/metabolismo , Proteínas de la Membrana/genética , Fibras Ópticas , Reacción en Cadena de la Polimerasa , Resonancia por Plasmón de Superficie/instrumentación , Calibración , Factores de TiempoRESUMEN
DNA- and MNAzymes are nucleic acid-based enzymes (NAzymes), which infiltrated the otherwise protein-rich field of enzymology three decades ago. The 10-23 core NAzymes are one of the most widely used and well-characterized NAzymes, but often require elevated working temperatures or additional complex modifications for implementation at standard room temperatures. Here, we present a generally applicable method, based on thermodynamic principles governing hybridization, to re-engineer the existing 10-23 core NAzymes for use at 23 °C. To establish this, we first assessed the activity of conventional NAzymes in the presence of cleavable and non-cleavable substrate at 23 °C as well as over a temperature gradient. These tests pointed towards a non-catalytic mechanism of signal generation at 23 °C, suggesting that conventional NAzymes are not suited for use at this temperature. Following this, several novel NAzyme-substrate complexes were re-engineered from the conventional ones and screened for their performance at 23 °C. The complex with substrate and substrate-binding arms of the NAzymes shortened by four nucleotides on each terminus demonstrated efficient catalytic activity at 23 °C. This has been further validated over a dilution of enzymes or enzyme components, revealing their superior performance at 23 °C compared to the conventional 10-23 core NAzymes at their standard operating temperature of 55 °C. Finally, the proposed approach was applied to successfully re-engineer three other new MNAzymes for activity at 23 °C. As such, these re-engineered NAzymes present a remarkable addition to the field by further widening the diverse repertoire of NAzyme applications.
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ADN Catalítico/química , ADN Catalítico/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , ADN/química , Ingeniería de Proteínas , Catálisis , Reproducibilidad de los Resultados , Especificidad por Sustrato , Temperatura , TermodinámicaRESUMEN
In this work, we present a new iSIMPLE concept (infusion Self-powered Imbibing Microfluidic Pump by Liquid Encapsulation), which requires no external power for activation nor liquid manipulation, it is easy to use while its fabrication method is extremely simple, inexpensive and suited for mass replication. The pump consists of a working liquid, which is - after finger activation - absorbed in a porous material (e.g. filter paper). The air expelled from the porous material increases the pressure in the downstream outlet channel and propels the outlet liquid (i.e. the sample) through the channel or ejects it. Here we investigated the influence of different filter papers on the iSIMPLE flow rates, achieving a wide range from 30 down to 0.07 µL/min. We also demonstrated the versatility of the iSIMPLE in terms of the liquid volume that can be manipulated (from 0.5 µL up to 150 µL) and the working pressure reaching 64 kPa, unprecedented high for a self-powered microfluidics pump. In addition, using a 34 G microneedle mounted on the iSIMPLE, we successfully injected liquids with different viscosities (from 0.93 up to 55.88 cP) both into an agarose matrix and a skin-like biological ex vivo substrate (i.e. chicken breast tissue). This work validated the compatibility of the iSIMPLE with drug delivery in a controlled way into a skin-like matrix, envisioning a whole new scenario for intradermal injections using self-contained skin patch. In addition, due to the extreme flexibility of the design and manufacturing, the iSIMPLE concept offers enormous opportunities for completely autonomous, portable and cost effective LOC devices.
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Sistemas de Liberación de Medicamentos/instrumentación , Bombas de Infusión , Dispositivos Laboratorio en un Chip , Filtración , Agujas , Papel , PresiónRESUMEN
Monitoring the concentration of a therapeutic drug antibody, infliximab (IFX), is recommended for enhancing its efficacy in patients with inflammatory bowel disease (IBD). However, IFX concentrations are currently determined in patients' serum/plasma, which requires sample preparation from blood, hence hampering the turnaround time. In this paper, we present a short immunoassay (10 min) using a fiber-optic surface plasmon resonance (FO-SPR) biosensor for detection of IFX spiked in 100-fold diluted serum, plasma, and whole blood. The calculated limits of detection (LOD) based on calibration curves were 1.42, 1.00, and 1.34 ng/mL, respectively, which coincides with expected IFX concentrations in diluted samples from IBD patients. A linear correlation was established among different matrixes, indicating that the matrix effect was insignificant. The established point-of-care (POC) FO-SPR bioassay was also used to measure IFX in 100-fold diluted extracts of dried blood spots (DBS), and LOD achieved was below 2 ng/mL. Although DBS might be ideal for POC, this is the first report of using an SPR biosensor for measuring DBS samples. Finally, the POC FO-SPR immunoassay was validated by using matching serum and plasma samples from five IBD patients. A Pearson correlation of 0.968 was obtained between serum and plasma samples. IFX concentrations determined with FO-SPR were compared to a clinically validated enzyme-linked immunosorbent assay (ELISA), resulting in excellent Pearson correlation and intraclass correlation coefficient, both being 0.99 for serum and plasma samples. In conclusion, this paper demonstrates that our FO-SPR biosensor can be used as a true POC diagnostic tool for determining IFX concentrations in a variety of matrixes.
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Técnicas Biosensibles , Ensayo de Inmunoadsorción Enzimática , Tecnología de Fibra Óptica , Enfermedades Inflamatorias del Intestino/sangre , Infliximab/sangre , Resonancia por Plasmón de Superficie , Calibración , Humanos , Límite de Detección , Sistemas de Atención de PuntoRESUMEN
Merging surface plasmon resonance (SPR) to fiber optic (FO) technology has brought remarkable achievements in the field by offering attractive advantages over the conventional prism-based SPR platforms, such as simplicity, cost-effectiveness and miniaturization. However, the performance of the existing FO-SPR instruments mainly depends on the device surface condition and in particular on the structural aspect of the thin gold (Au) plasmonic film deposited on the FO substrate. In this work, a simple cost-effective colloidal lithography technique (CLT) was adapted and applied for the first time to the micrometer-sized FO substrate, to design end reflection-type FO-SPR sensors with periodic arrays of Au triangularly-shaped nanostructures on the Au mirror FO tip distal end. The nanopatterned FO-SPR sensor tips were afterwards subjected to refractometric measurements in a sucrose dilution series and subsequently compared with their non-patterned counterparts. It was observed that the spectral dips of the nanopatterned FO-SPR sensor tips were shifted towards longer wavelengths after CLT patterning. Moreover, the sensor sensitivity was improved with up to 25% compared to the conventional non-patterned FO-SPR devices. The obtained results represent important steps in the development of a new generation of FO-SPR sensors with improved performance, which can ultimately be used in various applications, ranging from food analysis and environmental monitoring, to health control and medical diagnosis.
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Abstract: Accurate identification and quantification of allergens is key in healthcare, biotechnology and food quality and safety. Celery (Apium graveolens) is one of the most important elicitors of food allergic reactions in Europe. Currently, the golden standards to identify, quantify and discriminate celery in a biological sample are immunoassays and two-step molecular detection assays in which quantitative PCR (qPCR) is followed by a high-resolution melting analysis (HRM). In order to provide a DNA-based, rapid and simple detection method suitable for one-step quantification, a fiber optic PCR melting assay (FO-PCR-MA) was developed to determine different concentrations of celery DNA (1 pM-0.1 fM). The presented method is based on the hybridization and melting of DNA-coated gold nanoparticles to the FO sensor surface in the presence of the target gene (mannitol dehydrogenase, Mtd). The concept was not only able to reveal the presence of celery DNA, but also allowed for the cycle-to-cycle quantification of the target sequence through melting analysis. Furthermore, the developed bioassay was benchmarked against qPCR followed by HRM, showing excellent agreement (R² = 0.96). In conclusion, this innovative and sensitive diagnostic test could further improve food quality control and thus have a large impact on allergen induced healthcare problems.
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The detection of single molecules in magnetic microbead microwell array formats revolutionized the development of digital bioassays. However, retrieval of individual magnetic beads from these arrays has not been realized until now despite having great potential for studying captured targets at the individual level. In this paper, optical tweezers were implemented on a digital microfluidic platform for accurate manipulation of single magnetic beads seeded in a microwell array. Successful optical trapping of magnetic beads was found to be dependent on Brownian motion of the beads, suggesting a 99% chance of trapping a vibrating bead. A tailor-made experimental design was used to screen the effect of bead type, ionic buffer strength, surfactant type, and concentration on the Brownian activity of beads in microwells. With the optimal conditions, the manipulation of magnetic beads was demonstrated by their trapping, retrieving, transporting, and repositioning to a desired microwell on the array. The presented platform combines the strengths of digital microfluidics, digital bioassays, and optical tweezers, resulting in a powerful dynamic microwell array system for single molecule and single cell studies.
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Técnicas Analíticas Microfluídicas , Pinzas Ópticas , Campos Magnéticos , Técnicas Analíticas Microfluídicas/instrumentación , Estreptavidina/químicaRESUMEN
Flu is caused by the influenza virus that, due to mutations, keeps our body vulnerable for infections, making early diagnosis essential. Although immuno-based diagnostic tests are available, they have low sensitivity and reproducibility. In this paper, the prospect of detecting influenza A virus using digital ELISA has been studied. To appropriately select bioreceptors for this bioassay, seven commercial antibodies against influenza A nucleoprotein were methodically tested for their reactivity and binding affinity. The study has been performed on two markedly different platforms, being an enzyme-linked immunosorbent assay and a surface plasmon resonance system. The selected antibodies displayed completely different behavior on the two platforms and in various assay configurations. Surprisingly, the antibodies that showed overall good reactivity on both platforms had the highest dissociation constant among the tested antibodies, suggesting that, although important, binding affinity is not the only parameter to be considered when selecting antibodies. Moreover, only one antibody had the capacity to capture the nucleoprotein directly in lysis buffer used for releasing this viral protein, which might pose a huge advantage when developing assays with a fast time-to-result. This antibody was implemented on an in-house developed digital ELISA platform for ultrasensitive detection of recombinant nucleoprotein, reaching a detection limit of 4 ± 1 fM in buffer and 10 ± 2 fM in 10-fold diluted nasopharyngeal swabs, which is comparable to currently available fast molecular detection techniques. These results point to a great potential for ultrasensitive immuno-based influenza detection.
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Ensayo de Inmunoadsorción Enzimática/métodos , Virus de la Influenza A/química , Proteínas de Unión al ARN/análisis , Proteínas del Núcleo Viral/análisis , Proteínas de la Nucleocápside , Proteínas Recombinantes/análisisRESUMEN
Aptamers are emerging as powerful synthetic bioreceptors for fundamental research, diagnostics, and therapeutics. For further advances, it is important to gain a better understanding of how aptamers interact with their targets. In this work, we have used magnetic force-induced dissociation experiments to study the dissociation process of two different aptamer-protein complexes, namely for hIgE and Ara h 1. The measurements show that both complexes exhibit dissociation with two distinct regimes: the dissociation rate depends weakly on the applied force at high forces but depends stronger on force at low forces. We attribute these observations to the existence of at least one intermediate state and at least two energy barriers in the aptamer-protein interaction. The measured spontaneous dissociation rate constants were validated with SPR using both Biacore and fiber optic technology. This work demonstrates the potential of the magnetic force-induced dissociation approach for an in-depth study of the dissociation kinetics of aptamer-protein bonds, which is not possible with SPR technologies. The results will help in the development and expansion of aptamers as bioaffinity probes.
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Aptámeros de Nucleótidos/química , Proteínas/química , Secuencia de Bases , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
Detection of single nucleotide polymorphisms (SNPs) is a crucial challenge in the development of a novel generation of diagnostic tools. Accurate detection of SNPs can prove elusive, as the impact of a single variable nucleotide on the properties of a target sequence is limited, even if this sequence consists of only a few nucleotides. New, accurate and facile strategies for the detection of point mutations are therefore absolutely necessary for the increased adoption of point-of-care molecular diagnostics. Currently, PCR and sequencing are mostly applied for diagnosing SNPs. However these methods have serious drawbacks as routine diagnostic tools because of their labour intensity and cost. Several new, more suitable methods can be applied to enable sensitive detection of mutations based on specially designed hybridization probes, mutation recognizing enzymes and thermal denaturation. Here, an overview is presented of the most recent advances in the field of fast and sensitive SNP detection assays with strong potential for integration in point-of-care tests.
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Hibridación de Ácido Nucleico/métodos , Polimorfismo de Nucleótido Simple , Humanos , Desnaturalización de Ácido Nucleico , Proteínas/metabolismo , TemperaturaRESUMEN
Self-powered microfluidics presents a revolutionary approach to address the challenges of healthcare in decentralized and point-of-care settings where limited access to resources and infrastructure prevails or rapid clinical decision-making is critical. These microfluidic systems exploit physical and chemical phenomena, such as capillary forces and surface tension, to manipulate tiny volumes of fluids without the need for external power sources, making them cost-effective and highly portable. Recent technological advancements have demonstrated the ability to preprogram complex multistep liquid operations within the microfluidic circuit of these standalone systems, which enabled the integration of sensitive detection and readout principles. This chapter first addresses how the accessibility to in vitro diagnostics can be improved by shifting toward decentralized approaches like remote microsampling and point-of-care testing. Next, the crucial role of self-powered microfluidic technologies to enable this patient-centric healthcare transition is emphasized using various state-of-the-art examples, with a primary focus on applications related to biofluid collection and the detection of either proteins or nucleic acids. This chapter concludes with a summary of the main findings and our vision of the future perspectives in the field of self-powered microfluidic technologies and their use for in vitro diagnostics applications.
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Técnicas Analíticas Microfluídicas , Ácidos Nucleicos , Sistemas de Atención de Punto , Proteínas , Humanos , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Microfluídica/instrumentación , Ácidos Nucleicos/análisis , Pruebas en el Punto de Atención , Proteínas/análisisRESUMEN
The day-old male chick culling remains a welfare issue in the poultry industry. Several governments have prohibited this practice, pushing hatcheries to seek alternatives. Although different solutions exist for solving this problem, sex determination during the embryo's incubation (in ovo sexing) is considered the most suitable one among the consumers and industry. However, to be industrialized, in ovo sexing technologies must meet several requirements: compatibility with all egg colors and early developmental stages while maintaining a high hatchability rate and accuracy at low cost and high throughput. To meet these requirements, we studied the use of the sexual genes HINTW (female-specific) and DMRT-1 (both sexes) at incubation days 6-9. By utilizing the quantitative polymerase chain reaction in allantoic fluid (AF) samples, our study confirmed female-specific HINTW detection on all days without any significant detrimental effects on embryo development. We achieved 95% sexing accuracy using the HINTW cycle threshold (Ct) alone and 100% accuracy rate when using Δλ values (difference between the HINTW and DMRT-1 Ct). In conclusion, the developed assay can provide information about AF as a sample for in ovo sexing and open new industrial possibilities for faster and cheaper assays.
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A home-built fiber optic surface plasmon resonance platform (FO-SPR) was applied to directly screen PCR amplified DNA for mutations. The FO-SPR sensor was used for real-time monitoring of DNA duplex melting during high resolution temperature cycling. The signal of the DNA melting was enhanced by means of gold nanoparticle labels. This FO-SPR genetic assay allowed for detection of single-point mutations (SNP) in less than 20 min. The concept was demonstrated for the analysis of 9 different serogroups of the bacterium Legionella pneumophila, a common human pathogen responsible for atypical pneumonia. FO-SPR allowed us to detect genetic mutations inhibiting PCR, which could lead to amplification bias when molecular diagnostics are applied for L. pneumophila detection. All serogroups were found to display unique melting temperatures, indicating that mutations have accumulated in the target sequence. In a next step, clinical samples of L. pneumophila were analyzed using the FO-SPR sensor. This technology was proven to be reliable for the detection of mutations for those samples that previously displayed ambiguous qPCR quantification results. When these results were benchmarked, FO-SPR results were found to be consistent with Sanger sequencing but not with fluorescence based DNA melting. The presented results convincingly advocate the advantages of FO-SPR as a high resolution and fast genetic screening tool that can compete with the current standard techniques for SNP detection.
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ADN Bacteriano/genética , Tecnología de Fibra Óptica/métodos , Legionella pneumophila/genética , Mutación/genética , Ácidos Nucleicos/genética , Resonancia por Plasmón de Superficie/métodos , Secuencia de Bases , ADN Bacteriano/aislamiento & purificación , Legionella pneumophila/aislamiento & purificación , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico/genética , Ácidos Nucleicos/aislamiento & purificaciónRESUMEN
Ever increasing demands in sensitivity and specificity of biosensors have recently established a trend toward the use of multivalent bioreceptors. This trend has also been introduced in the field of bacteriophage affinity peptides, where the entire phage is used as a receptor rather than the individual peptides. Although this approach is gaining in popularity due to the numerous advantages, binding kinetics of complete phage particles have never been studied in detail, notwithstanding being essential for the efficient design of such applications. In this paper we used an in house developed fiber-optic surface plasmon resonance (FO-SPR) biosensor to study the affinity and binding kinetics of phages, displaying peptide libraries. By using either peptide expression on the p3 or on the p8 coat proteins, a corresponding density of 5 up to more than 2000 peptides on a single virus particle was obtained. Binding parameters of 26 different filamentous phages, displaying peptides selective for enhanced Green Fluorescent Protein (eGFP), were characterized. This study revealed a broad affinity range of phages for the target eGFP, indicating their potential to be used for applications with different requirements in binding kinetics. Moreover, detailed analysis of koff and kon values of several selected p3 and p8 phages, using the FO-SPR biosensor, clearly showed the correlation between the binding parameters and the density at which eGFP-peptides are being expressed. Consequently, although p3 and p8-based phages both revealed exceptionally high affinities for eGFP, two p8 phages were found to have the highest affinity with dissociation constants (Kd) in the femtomolar range.
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Bacteriófagos/genética , Proteínas Portadoras/análisis , Péptidos/análisis , Resonancia por Plasmón de Superficie/métodos , Proteínas Portadoras/genética , Ensayo de Inmunoadsorción Enzimática , Proteínas Fluorescentes Verdes/química , Péptidos y Proteínas de Señalización Intercelular , Péptidos/genéticaRESUMEN
Major advancements in molecular biology and clinical diagnostics cannot be brought about strictly through the use of genomics based methods. Improved methods for protein detection and proteomic screening are an absolute necessity to complement to wealth of information offered by novel, high-throughput sequencing technologies. Only then will it be possible to advance insights into clinical processes and to characterize the importance of specific protein biomarkers for disease detection or the realization of "personalized medicine". Currently however, large-scale proteomic information is still not as easily obtained as its genomic counterpart, mainly because traditional antibody-based technologies struggle to meet the stringent sensitivity and throughput requirements that are required whereas mass-spectrometry based methods might be burdened by significant costs involved. However, recent years have seen the development of new biodetection strategies linking nucleic acids with existing antibody technology or replacing antibodies with oligonucleotide recognition elements altogether. These advancements have unlocked many new strategies to lower detection limits and dramatically increase throughput of protein detection assays. In this review, an overview of these new strategies will be given.
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Ácidos Nucleicos/química , Proteínas/análisis , Proteómica/métodos , Aptámeros de Nucleótidos/química , HumanosRESUMEN
Fluorescence in situ hybridization (FISH) is the gold standard for visualizing genomic DNA in fixed cells and tissues, but it is incompatible with live-cell imaging, and its combination with RNA imaging is challenging. Consequently, due to its capacity to bind double-stranded DNA (dsDNA) and design flexibility, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) technology has sparked enormous interest over the past decade. In this review, we describe various nucleic acid (NA)- and protein-based (amplified) signal generation methods that achieve imaging of repetitive and single-copy sequences, and even single-nucleotide variants (SNVs), next to highly multiplexed as well as dynamic imaging in live cells. With future progress in the field, the CRISPR-(d)Cas9-based technology promises to break through as a next-generation cell-imaging technique.