RESUMEN

A biocompatible, highly sensitive, and enzyme-free glucose electrochemical sensor was developed based on a copper-cysteamine (Cu-Cy)-modified electrode. The catalytically active biocompatible material Cu-Cy was immobilized on the electrode surface by the natural polymer chitosan (CTS). The electrochemical characterization and glucose response of the Cu-Cy/CTS/glassy carbon electrode (GCE) were investigated by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and constant potential amperometry. The significant electrocatalytic activity of Cu-Cy to the oxidation of glucose in an alkaline environment was revealed. Several crucial parameters, including the number of scanning cycles for electrode activation, applied potential, and the contents of Cu-Cy and chitosan, were investigated to understand their impact on the sensor's response. The proposed sensing platform exhibited linear ranges of 2.7 µM to 1.3 mM and 1.3 mM to 7.7 mM for glucose detection, coupled with high sensitivity (588.28 and 124.42 µA·mM-1·cm-2), and commendable selectivity and stability. Moreover, a Cu-Cy/CTS-modified screen-printed electrode (SPE) was further developed for portable direct detection of glucose in real samples.

RESUMEN

BACKGROUND: Measurement of endogenous cellular hydrogen peroxide (H2O2) can provide information on cellular status, and help to understand cellular metabolism and signaling processes, thus contributing to elucidation of disease mechanisms and new diagnostics/therapeutic approaches. RESULTS: In this work, Pt-Cd bimetallic nanozyme was successfully prepared via the solvothermal synthetic method for sensitive detection of H2O2. The synthesized Pt-Cd bimetallic nanozyme could exhibited good electrochemical activity. Then, the materials were analyzed for the electrochemical properties and catalytic properties of H2O2 by cyclic voltammetry and chronoamperometry, respectively. Results indicated that the synthesized nanozyme had superior sensitivity (295 µA⸳mM-1⸳cm-2) and selectivity toward H2O2 with a detection limit of 0.21 µM. Further, the Pt-Cd bimetallic nanozyme displayed good electrochemical properties compared to platinum catalysts alone. The application was extended to determine the produced H2O2 from human hepatocellular carcinoma cells (HepG2) and normal hepatocyte (LO2) samples after ascorbic acid stimulation, thus enabling the early warning of cellular carcinogenesis. SIGNIFICANCE: This strategy promises simple, rapid, inexpensive and effective electrochemical sensing and provides a new pathway for the synthesis of bimetallic nanozymes to construct an electrochemical sensor for the sensitive detection of H2O2.

Asunto(s)

RESUMEN

Reliable and cost-effective glucose sensors are in rising demand among diabetes patients. The combination of metals and conducting polymers creates a robust electrocatalyst for glucose oxidation, offering enzyme-free, high stability, and sensitivity with outstanding electrochemical results. Herein, graphene is grown on nickel foam by chemical vapor deposition to make a graphene@nickel foam scaffold (G@NF), on which silver nanoplates-polyaniline (Ag-PANI) 3D architecture is developed by sonication-assisted co-electrodeposition. The resulting binder-free 3D Ag-PANI/G@NF electrode was highly porous, as characterized by XPS, FESEM, XRD, FTIR, and Raman spectroscopy. The binder-free 3D Ag-PANI/G@NF electrode exhibits remarkable electrochemical efficiency with a superior electrochemical active surface area. The amperometric analysis provides excellent anti-interference performance, a low limit of deduction (0.1 nM), robust sensitivity (1.7 x 1013 µA mM-1cm-2), and a good response time. Moreover, the Ag-PANI/G@NF enzyme-free sensor is utilized to observe glucose levels in human blood serums and exhibits excellent potential to become a reliable clinical glucose sensor.

RESUMEN

In the field of nucleic acid amplification assays, developing enzyme-free, easy-to-use, and highly sensitive amplification approaches remains a challenge. In this work, we synthesized a heterogeneous Cu2O nanocatalyst (hnCu2O) with different particle sizes and shapes, which was used for developing enzyme- and label-free nucleic acid amplification methods based on the nucleic acid-templated azide-alkyne cycloaddition (AAC) reaction catalyzed by hnCu2O. The hnCu2O exhibited size- and shape-dependent catalytic activity, with smaller sizes and spherical-like shapes exhibiting superior activity. Spherical-like hnCu2O (61 ± 8 nm) not only achieved a ligation yield of up to 84.2 ± 3.9 % in 3 min but also exhibited faster kinetics in the nucleic acid-templated hnCu2O-catalyzed AAC reaction, with a high reaction rate of 0.65 min-1 and a half-life of 1.07 ± 0.09 min. Based on this result, we developed nucleic acid-templated click ligation linear amplification reaction (NA-CLLAR) and nucleic acid-templated click ligation exponential amplification reaction (NA-CLEAR) approach. By combining the recognition (complementary to the target sequence) and signal output (split G-quadruplex sequence) elements into a DNA probe, the NA-CLLAR and NA-CLEAR fluorescence assays achieved highly specific detection of target nucleic acids, with a detection limit of 2.8 aM based on G-quadruplex-enhanced fluorescence. This work is a valuable reference and will inspire researchers to design enzyme-free nucleic acid signal amplification strategies by developing different types of Cu(I) catalysts with improved catalytic activity.

Asunto(s)

RESUMEN

A sandwich-type electrochemical immunosensor was proposed for the ultra-sensitive detection of CD44, a potential biomarker for breast cancer. In this design, a customized template-based ionic liquid (1-butyl-2,3-dimethylimidazolium tetrafluoroborate) carbon paste electrode (CILE) served as the sensing platform, and thionine/Au nanoparticles/covalent-organic frameworks (THI/Au/COF) were used as the signal label. Moreover, an enzyme-free signal amplification strategy was introduced by involving H2O2 and phosphotungstate (PW12) with peroxidase-like activity. Under optimized conditions, the linear range is as wide as six orders of magnitude, and the detection limit is as low as 0.71 pg mL-1 (estimated based on S/N = 3). Average recoveries range from 98.16 %-100.1 %, with a relative standard deviation (RSD) of 1.42-8.27 % in mouse serum, and from 98.44 %-99.06 %, with an RSD of 1.14-4.84 % (n = 3) in artificial saliva. Furthermore, the immunosensor exhibits excellent specificity toward CD44, good stability, and low cost, indicating great potential for application in clinical trials.

Asunto(s)

RESUMEN

Microphysiological systems have attracted attention because of their use in drug screening. However, it is challenging to measure cell functions in real time using a device. In this study, we developed a cell culture device using a porous membrane electrode for in situ electrochemical glucose measurements for cell analysis. First, a porous membrane electrode was fabricated and electrochemically evaluated for enzyme-free glucose measurement. Subsequently, the glucose uptake of MCF-7 spheroids was evaluated using living spheroids, fixed spheroids, supernatants, and glucose transporter inhibitor-treated spheroids. Conventionally, the direct optical measurement of glucose uptake requires fluorescence-labeled glucose derivatives. In addition, the glucose uptake can be evaluated by measuring the glucose concentration in the medium by optical or electrochemical measurements. However, glucose needs to be consumed in the entire cell culture medium, which needs a long culture time. In contrast, our system can measure glucose in approximately 5 min without any labels because of in situ electrochemical measurements. This system can be used for in situ measurements in in vitro cell culture systems, including organ-on-a-chip for drug screening.

Asunto(s)

RESUMEN

In this work, a new photoelectrochemical (PEC) biosensor based on triple quenching effect of nanozyme catalyzed precipitation to PEC signal of MgIn2S4 was constructed for ultrasensitive detection of circulating tumor DNA (ctDNA). Enzyme-free amplification technology was used to convert target ctDNA into a large number of product chains (PC) to improve the detection sensitivity. Co3O4 nanozyme with excellent peroxidase (POD)-like activity was introduced to the surface of MgIn2S4 by PC. Co3O4 could oxidize chromogenic agent 3-Amino-9-ethylcarbazole (AEC) to produce red insoluble precipitation in the presence of H2O2, resulting in the PEC signal "off" of MgIn2S4 to achieve ultrasensitive detection of ctDNA. In particular, Co3O4 nanozyme showed three synergistic quenching effects on PEC signal of MgIn2S4, which contributed greatly to improving the detection sensitivity. Firstly, the light absorption range of Co3O4 could reach 1000 nm, and compete with MgIn2S4 for light absorption. Secondly, the produced red precipitation belonged to the insulating material and had large electrochemical impedance, which hindered the transmission of photogenerated carriers. Thirdly, the precipitation also prevented the electron donor ascorbic acid (AA) from transferring electrons to MgIn2S4. This biosensor provided a promising sensitive PEC detection technology for ctDNA, and further broadened the application of nanozymes in the field of PEC analysis.

Asunto(s)

RESUMEN

The development of sensitive and efficient analytical methods for multiple biomarkers is crucial for cancer screening at early stage. MicroRNAs (miRNAs) are a kind of biomarkers with diagnostic potential for cancer. However, the ultrasensitive and logical analysis of multiple miRNAs with simple operation still faces some challenges. Herein, a photonic crystal (PC)-enhanced fluorescence biosensor with logic gate operation based on one-pot cascade amplification DNA circuit was developed for enzyme-free and ultrasensitive analysis of two cancer-related miRNAs. The fluorescence biosensor was performed by biochemical recognition amplification module (BCRAM) and physical enhancement module (PEM) to achieve logical and sensitive detection. In the BCRAM, one-pot cascade amplification circuit consisted of the upstream parallel entropy-driven circuit (EDC) and the downstream shared catalytic hairpin assembly (CHA). The input of target miRNA would trigger each corresponding EDC, and the parallel EDCs released the same R strand for triggering subsequent CHA; thus, the OR logic gate was obtained with minimization of design and operation. In the PEM, photonic crystal (PC) array was prepared easily for specifically enhancing the fluorescence output from BCRAM by the optical modulation capabilities; meanwhile, the high-throughput signal readout was achieved by microplate analyzer. Benefiting from the integrated advantages of two modules, the proposed biosensor achieved ultrasensitive detection of two miRNAs with easy logic gate operation, obtaining the LODs of 8.6 fM and 6.7 fM under isothermal and enzyme-free conditions. Hence, the biosensor has the advantages of high sensitivity, easy operation, multiplex and high-throughput analysis, showing great potential for cancer screening at early stage.

Asunto(s)

RESUMEN

Single-nucleotide variants (SNVs) are the most common type variation of sequence alterations at a specific location in the genome, thus involving significant clinical and biological information. The assay of SNVs has engaged great awareness, because many genome-wide association studies demonstrated that SNVs are highly associated with serious human diseases. Moreover, the investigation of SNV expression levels in single cells are capable of visualizing genetic information and revealing the complexity and heterogeneity of single-nucleotide mutation-related diseases. Thus, developing SNV assay approaches in vitro, particularly in single cells, is becoming increasingly in demand. In this review, we summarized recent progress in the enzyme-free and enzyme-mediated strategies enabling SNV assay transition from sensing interface to the test tube and single cells, which will potentially delve deeper into the knowledge of SNV functions and disease associations, as well as discovering new pathways to diagnose and treat diseases based on individual genetic profiles. The leap of SNV assay achievements will motivate observation and measurement genetic variations in single cells, even within living organisms, delve into the knowledge of SNV functions and disease associations, as well as open up entirely new avenues in the diagnosis and treatment of diseases based on individual genetic profiles.

RESUMEN

Today, diabetes mellitus is one of the most common diseases that affects the population on a worldwide scale. Patients suffering from this disease are required to control their blood-glucose levels several times a day through invasive methods such as piercing their fingers. Our NaGdF4: 5% Er3+, 3% Nd3+ nanoparticles demonstrate a remarkable ability to detect D-glucose levels by analysing alterations in their red-to-green ratio, since this sensitivity arises from the interaction between the nanoparticles and the OH groups present in the D-glucose molecules, resulting in discernible changes in the emission of the green and red bands. These luminescent sensors were implemented and tested on paper substrates, offering a portable, low-cost and enzyme-free solution for D-glucose detection in aqueous solutions with a limit of detection of 22 mg/dL. With this, our study contributes to the development of non-invasive D-glucose sensors, holding promising implications for managing diabetes and improving overall patient well-being with possible future applications in D-glucose sensing through tear fluid.

Asunto(s)

RESUMEN

The enhancement of detection sensitivity in microfluidic sensors has been a continuously explored field. Initially, many strategies for sensitivity improvement involved introducing enzyme cascade reactions, but enzyme-based reactions posed challenges in terms of cost, stability, and storage. Therefore, there is an urgent need to explore enzyme-free cascade amplification methods, which are crucial for expanding the application range and improving detection stability. Metal or metal compound nanomaterials have gained great attention in the exploitation of microfluidic sensors due to their ease of preparation, storage, and lower cost. The unique physical properties of metallic nanomaterials, including surface plasmon resonance, surface-enhanced Raman scattering, metal-enhanced fluorescence, and surface-enhanced infrared absorption, contribute significantly to enhancing detection capabilities. The metal-based catalytic nanomaterials, exemplified by Fe3O4 nanoparticles and metal-organic frameworks, are considered viable alternatives to biological enzymes due to their excellent performance. Herein, we provide a detailed overview of the applications of metals and metal compounds in improving the sensitivity of microfluidic biosensors. This review not only highlights the current developments but also critically analyzes the challenges encountered in this field. Furthermore, it outlines potential directions for future research, contributing to the ongoing development of microfluidic biosensors with improved detection sensitivity.

Asunto(s)

RESUMEN

Obtaining high-quality omics data at the single-cell level from archived human tissue samples is crucial for gaining insights into cellular heterogeneity and pushing the field of personalized medicine forward. In this technical brief we present a comprehensive methodological framework for the efficient enzyme-free preparation of tissue-derived single cell suspensions and their conversion into single-cell miRNA sequencing libraries. The resulting data from this study have the potential to deepen our understanding of miRNA expression at the single-cell level and its relevance in the context of the examined tissues. The workflow encompasses tissue collection, RNALater immersion, storage, thawing, TissueGrinder-mediated dissociation, miRNA lysis, library preparation, sequencing, and data analysis. Quality control measures ensure reliable miRNA data, with specific attention to sample quality. The UMAP analysis reveals tissue-specific cell clustering, while miRNA diversity reflects tissue variations. The presented workflow effectively processes preserved tissues, extending opportunities for retrospective analysis and biobank utilization.

Asunto(s)

RESUMEN

In this study, we present an enzyme-free fluorescence biosensor for the highly sensitive detection of miRNA-21, a crucial biomarker in clinical diagnosis. Our innovative approach combines catalytic hairpin assembly (CHA) and entropy-driven amplification into a cascade amplification strategy. MicroRNA initiates the catalytic hairpin assembly reaction, liberating the trigger region needed for the entropy-driven amplification reaction. This triggers a series of strand displacement reactions, resulting in the separation of the fluorescence resonance energy transfer pair and an amplified fluorescence signal from FAM. Our cascade amplification strategy achieves ultra-sensitive microRNA detection, with an impressive limit of detection (LOD) of 1.3 fM, approximately 100-fold lower than CHA alone. Additionally, we successfully applied this biosensor for microRNA quantification in human serum and cell lysates, demonstrating its practicality and potential for early diagnosis.

Asunto(s)

RESUMEN

Point of care (PoC) nucleic acid amplification tests (NAATs) are a cornerstone of public health, providing the earliest and most accurate diagnostic method for many communicable diseases, such as HIV, in the same location the patient receives treatment. Communicable diseases disproportionately impact low-resource communities where NAATs are often unobtainable due to the resource intensive enzymes that drive the tests. Enzyme-free nucleic acid detection methods, such as hybridization chain reaction (HCR), use DNA secondary structures for self-driven amplification schemes producing large DNA nanostructures and capable of single molecule detection in cellulo. These thermodynamically driven DNA-based tests have struggled to penetrate the PoC diagnostic field due to their inadequate limits of detection or complex workflows. Here we present a proof-of-concept NAAT that combines HCR-based amplification of a target nucleic acid sequence with paper-based nucleic acid filtration and enrichment capable of detecting sub pM levels of synthetic DNA. We reconstruct the favorable hybridization conditions of an in cellulo reaction in vitro by incubating HCR in an evaporating, microvolume environment containing poly(ethylene glycol) as a crowding agent. We demonstrate that the kinetics and thermodynamics of DNA-DNA and DNA-RNA hybridization is enhanced by the dynamic evaporating environment and inclusion of crowding agents, bringing HCR closer to meeting PoC NAAT needs.

RESUMEN

Here, iron chalcogenide thin films were developed for the first time by using the less hazardous electrodeposition technique at optimized conditions on an FTO glass substrate. The chalcogenides have different surface, morphological, structural, and optical properties, as well as an enzyme-free sensing behavior toward urea. Numerous small crystallites of about ∼20 to 25 nm for FeSe, ∼18 to 25 nm for FeTe, and ∼18 to 22 nm in diameter for FeSeTe are observed with partial agglomeration under an electron microscope, having a mixed phase of tetragonal and orthorhombic structures of FeSe, FeTe, and, FeSeTe, respectively. Profilometry, XRD, FE-SEM, HR-TEM, XPS, EDX, UV-vis spectroscopy, and FT-IR spectroscopy were used for the analysis of binary and ternary composite semiconductors, FeSe, FeTe, and FeSeTe, respectively. Electrochemical experiments were conducted with the chalcogenide thin films and urea as the analyte in phosphate-buffered media at a pH of ∼ 7.4 in the concentration range of 3-413 µM. Cyclic voltammetry was performed to determine the sensitivity of the prepared electrode at an optimized scan rate of 50 mV s-1. The electrodeposited chalcogenide films appeared with a low detection limit and satisfactory sensitivity, of which the ternary chalcogenide film has the lowest LOD of 1.16 µM and the maximum sensitivity of 74.22 µA µM-1 cm-2. The transition metal electrode has a very wide range of detection limit of 1.25-2400 µM with a short response time of 4 s. This fabricated biosensor is capable of exhibiting almost 75% of its starting activity after 2 weeks of storage in the freezer at 4 °C. Simple methods of preparation, a cost-effective process, and adequate electrochemical sensing of urea confirm that the prepared sensor is suitable as an enzyme-free urea sensor and can be utilized for future studies.

Asunto(s)

RESUMEN

An innovative electrochemical sensor is introduced that utilizes bipolar electrochemistry on a paper substrate for detecting glucose in sweat. The sensor employs a three-dimensional porous nanocomposite (MXene/NiSm-LDH) formed by decorating nickel-samarium nanoparticles with double-layer MXene hydroxide. These specially designed electrodes exhibit exceptional electrocatalytic activity during glucose oxidation. The glucose sensing mechanism involves enzyme-free oxidation of the analyte within the sensor cell, achieved by applying an appropriate potential. This leads to the reduction of K3Fe(CN)6 in the reporter cell, and the resulting current serves as the response signal. By optimizing various parameters, the measurement platform enables the accurate determination of sweat glucose concentrations within a linear range of 10 to 200 µM. The limit of detection (LOD) for glucose is 3.6 µM (S/N = 3), indicating a sensitive and reliable detection capability. Real samples were analysed  to validate the sensor's efficiency, and the results obtained were both promising and encouraging.

Asunto(s)

RESUMEN

Primary cells form the basis of modern-day in vitro research analysis tools. Many conventional procedures for generating single-cell suspensions from solid tissue are neither robust nor reproducible. Here we describe primary cells isolation from surgically resected tumor tissue via enzyme-free mechanical dissociation using TissueGrinder, a novel semi-automated benchtop device. The isolated cells can be used for any downstream biochemical or cell-based analytic assay.

Asunto(s)

RESUMEN

A heterogeneous sensitive microRNA-155 assay based on a new isothermal amplification method, called catalytic hairpin assembly with oligonucleotide release (CHAOR), was developed. The principle of CHAOR was studied by non-denaturing electrophoresis. To detect the amplification product, a polyperoxidase-streptavidin conjugate (molar ratio 1:80) and an enhanced chemiluminescence reaction were used, which made it possible to increase assay sensitivity. The detection limit of microRNA-155 assay was 0.4 pM. The coefficient of variation of the chemiluminescent signal, formed upon the heterogeneous determination of miRNA-155, was less than 12 % within the working range. The efficiency of CHAOR as an amplification method was similar to that of traditional CHA, as miRNA-155 assays based on CHAOR and CHA had similar analytical parameters. In addition, the proposed assay was highly specific. Contrary to traditional CHA, CHAOR, one of whose products is a single-stranded oligonucleotide, can be used in analytical methods based on cascade amplification.

Asunto(s)

RESUMEN

The rapid and accurate detection of miRNAs is of great significance for early diagnosis and treatment of cancer. Hence, a novel enzyme-free and label-free electrochemical biosensor based on bio-barcode amplification for detecting miRNAs was presented. Sandwich structures constructed of magnetic nanoparticles modified with DNA probes, gold nanoparticles with numerous barcoded DNA strands that hybridized with target miRNAs were fabricated as the amplifier. The released barcoded DNA strands then acted as the secondary targets and triggered the electrochemical sensor with a significant electrochemical response. A highly sensitive (detection limit of 0.24 fM) and selective electrochemical miRNA detection was realized, which has great potential for application in miRNA-related clinical diagnosis and biochemical research.

Asunto(s)

RESUMEN

Here, an enzyme-free lateral flow aptasensor was designed by target-induced strand-displacement effect and followed by the activation of multi-component nucleic acid enzyme (MNAzyme)-mediated cleavage to enable rapid and portable ochratoxin A (OTA) detection. The substrate was prepared as an oligonucleotide strand modified with magnetic beads (MB) and human chorionic gonadotropin (hCG). The interaction of OTA with the aptamer induces the release of blocking DNA, which hybridized with three separated subunits of DNA, forming a sequence-specific MNAzyme catalytic core. This core subsequently initiated an enzyme-free MNAzyme cleavage reaction in the presence of the Mg2+ cofactor, cleaving a special substrate and releasing both the incomplete MNAzyme catalytic core and hCG-DNA probe. The incomplete MNAzyme catalytic core was then recognized by substrates once again, triggering a cascade recycling cleavage and resulting in the generation of a larger number of hCG-DNA probes. After magnetic enrichment, the free hCG-DNA probes flow through the pregnancy test strip (PTS) to the T line, generating a colorimetric readout that unequivocally confirms the presence of the target OTA. This work leverages the efficient enzyme-free cleavage amplification of MNAzyme and the PTS-based portable detection device, presenting a biosensing strategy with significant potential for sensitive and portable OTA detection. This method exhibited remarkable sensitivity and selectivity for OTA detection, boasting a detection limit of 5 nM. The present study successfully demonstrated the practical application of this method on real samples, offering a viable alternative for rapid and portable detection of mycotoxins.

Asunto(s)