Nanomotor-Enabled pH-Responsive Intracellular Delivery of Caspase-3: Toward Rapid Cell Apoptosis

Direct and efficient intracellular delivery of enzymes to cytosol holds tremendous therapeutic potential while remaining an unmet technical challenge. Herein, an ultrasound (US)-propelled nanomotor approach and a high-pH-responsive delivery strategy are reported to overcome this challenge using caspase-3 (CASP-3) as a model enzyme. Consisting of a gold nanowire (AuNW) motor with a pH-responsive polymer coating, in which the CASP-3 is loaded, the resulting nanomotor protects the enzyme from release and deactivation prior to reaching an intracellular environment. However, upon entering a cell and exposure to the higher intracellular pH, the polymer coating is dissolved, thereby directly releasing the active CASP-3 enzyme to the cytosol and causing rapid cell apoptosis. <i>In vitro</i> studies using gastric cancer cells as a model cell line demonstrate that such a motion-based active delivery approach leads to remarkably high apoptosis efficiency within a significantly shorter time and with a lower amount of CASP-3 compared to other control groups not involving US-propelled nanomotors. For instance, the reported nanomotor system can achieve 80% apoptosis of human gastric adenocarcinoma cells within only 5 min, which dramatically outperforms other CASP-3 delivery approaches. These results indicate that the US-propelled nanomotors may act as a powerful vehicle for cytosolic delivery of active therapeutic proteins, which would offer an attractive means to enhance the current landscape of intracellular protein delivery and therapy. While CASP-3 is selected as a model protein in this study, the same nanomotor approach can be readily applied to a variety of different therapeutic proteins

Nanomotor-Enabled pH-Responsive Intracellular Delivery of Caspase-3: Toward Rapid Cell Apoptosis

Direct and efficient intracellular delivery of enzymes to cytosol holds tremendous therapeutic potential while remaining an unmet technical challenge. Herein, an ultrasound (US)-propelled nanomotor approach and a high-pH-responsive delivery strategy are reported to overcome this challenge using caspase-3 (CASP-3) as a model enzyme. Consisting of a gold nanowire (AuNW) motor with a pH-responsive polymer coating, in which the CASP-3 is loaded, the resulting nanomotor protects the enzyme from release and deactivation prior to reaching an intracellular environment. However, upon entering a cell and exposure to the higher intracellular pH, the polymer coating is dissolved, thereby directly releasing the active CASP-3 enzyme to the cytosol and causing rapid cell apoptosis. <i>In vitro</i> studies using gastric cancer cells as a model cell line demonstrate that such a motion-based active delivery approach leads to remarkably high apoptosis efficiency within a significantly shorter time and with a lower amount of CASP-3 compared to other control groups not involving US-propelled nanomotors. For instance, the reported nanomotor system can achieve 80% apoptosis of human gastric adenocarcinoma cells within only 5 min, which dramatically outperforms other CASP-3 delivery approaches. These results indicate that the US-propelled nanomotors may act as a powerful vehicle for cytosolic delivery of active therapeutic proteins, which would offer an attractive means to enhance the current landscape of intracellular protein delivery and therapy. While CASP-3 is selected as a model protein in this study, the same nanomotor approach can be readily applied to a variety of different therapeutic proteins

Bacterial Isolation by Lectin-Modified Microengines

New template-based self-propelled gold/nickel/polyaniline/platinum (Au/Ni/PANI/Pt) microtubular engines, functionalized with the Concanavalin A (ConA) lectin bioreceptor, are shown to be extremely useful for the rapid, real-time isolation of <i>Escherichia coli</i> (<i>E. coli</i>) bacteria from fuel-enhanced environmental, food, and clinical samples. These multifunctional microtube engines combine the selective capture of <i>E. coli</i> with the uptake of polymeric drug-carrier particles to provide an attractive motion-based theranostics strategy. Triggered release of the captured bacteria is demonstrated by movement through a low-pH glycine-based dissociation solution. The smaller size of the new polymer-metal microengines offers convenient, direct, and label-free optical visualization of the captured bacteria and discrimination against nontarget cells

Bacterial Isolation by Lectin-Modified Microengines

New template-based self-propelled gold/nickel/polyaniline/platinum (Au/Ni/PANI/Pt) microtubular engines, functionalized with the Concanavalin A (ConA) lectin bioreceptor, are shown to be extremely useful for the rapid, real-time isolation of <i>Escherichia coli</i> (<i>E. coli</i>) bacteria from fuel-enhanced environmental, food, and clinical samples. These multifunctional microtube engines combine the selective capture of <i>E. coli</i> with the uptake of polymeric drug-carrier particles to provide an attractive motion-based theranostics strategy. Triggered release of the captured bacteria is demonstrated by movement through a low-pH glycine-based dissociation solution. The smaller size of the new polymer-metal microengines offers convenient, direct, and label-free optical visualization of the captured bacteria and discrimination against nontarget cells

Bacterial Isolation by Lectin-Modified Microengines

New template-based self-propelled gold/nickel/polyaniline/platinum (Au/Ni/PANI/Pt) microtubular engines, functionalized with the Concanavalin A (ConA) lectin bioreceptor, are shown to be extremely useful for the rapid, real-time isolation of <i>Escherichia coli</i> (<i>E. coli</i>) bacteria from fuel-enhanced environmental, food, and clinical samples. These multifunctional microtube engines combine the selective capture of <i>E. coli</i> with the uptake of polymeric drug-carrier particles to provide an attractive motion-based theranostics strategy. Triggered release of the captured bacteria is demonstrated by movement through a low-pH glycine-based dissociation solution. The smaller size of the new polymer-metal microengines offers convenient, direct, and label-free optical visualization of the captured bacteria and discrimination against nontarget cells

Bacterial Isolation by Lectin-Modified Microengines

New template-based self-propelled gold/nickel/polyaniline/platinum (Au/Ni/PANI/Pt) microtubular engines, functionalized with the Concanavalin A (ConA) lectin bioreceptor, are shown to be extremely useful for the rapid, real-time isolation of <i>Escherichia coli</i> (<i>E. coli</i>) bacteria from fuel-enhanced environmental, food, and clinical samples. These multifunctional microtube engines combine the selective capture of <i>E. coli</i> with the uptake of polymeric drug-carrier particles to provide an attractive motion-based theranostics strategy. Triggered release of the captured bacteria is demonstrated by movement through a low-pH glycine-based dissociation solution. The smaller size of the new polymer-metal microengines offers convenient, direct, and label-free optical visualization of the captured bacteria and discrimination against nontarget 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

Single Cell Real-Time miRNAs Sensing Based on Nanomotors

A nanomotor-based strategy for rapid single-step intracellular biosensing of a target miRNA, expressed in intact cancer cells, at the single cell level is described. The new concept relies on the use of ultrasound (US) propelled dye-labeled single-stranded DNA (ssDNA)/graphene-oxide (GO) coated gold nanowires (AuNWs) capable of penetrating intact cancer cells. Once the nanomotor is internalized into the cell, the quenched fluorescence signal (produced by the π–π interaction between GO and a dye-labeled ssDNA) is recovered due to the displacement of the dye-ssDNA probe from the motor GO-quenching surface upon binding with the target miRNA-21, leading to an attractive intracellular “OFF-ON” fluorescence switching. The faster internalization process of the US-powered nanomotors and their rapid movement into the cells increase the likelihood of probe–target contacts, leading to a highly efficient and rapid hybridization. The ability of the nanomotor-based method to screen cancer cells based on the endogenous content of the target miRNA has been demonstrated by measuring the fluorescence signal in two types of cancer cells (MCF-7 and HeLa) with significantly different miRNA-21 expression levels. This single-step, motor-based miRNAs sensing approach enables rapid “on the move” specific detection of the target miRNA-21, even in single cells with an extremely low endogenous miRNA-21 content, allowing precise and real-time monitoring of intracellular miRNA expression

Integrated Amperometric Affinity Biosensors Using Co²⁺–Tetradentate Nitrilotriacetic Acid Modified Disposable Carbon Electrodes: Application to the Determination of β‑Lactam Antibiotics

A novel strategy for the construction of disposable amperometric affinity biosensors is described in this work. The approach uses a recombinant bacterial penicillin binding protein (PBP) tagged by an N-terminal hexahistidine tail which was immobilized onto Co²⁺–tetradentate nitrilotriacetic acid (NTA)-modified screen-printed carbon electrodes (SPCEs). The biosensor was employed for the specific detection and quantification of β-lactam antibiotics residues in milk, which was accomplished by means of a direct competitive assay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling. The amperometric response measured at −0.20 V versus the Ag pseudoreference electrode of the SPCE upon the addition of H₂O₂ in the presence of hydroquinone (HQ) as redox mediator was used as the transduction signal. The developed affinity sensor allowed limits of detection to be obtained in the low part-per-billion level for the antibiotics tested in untreated milk samples. Moreover, the biosensor exhibited a good selectivity against other antibiotics residues frequently detected in milk and dairy products. The analysis time was of approximately 30 min

Single Cell Real-Time miRNAs Sensing Based on Nanomotors

A nanomotor-based strategy for rapid single-step intracellular biosensing of a target miRNA, expressed in intact cancer cells, at the single cell level is described. The new concept relies on the use of ultrasound (US) propelled dye-labeled single-stranded DNA (ssDNA)/graphene-oxide (GO) coated gold nanowires (AuNWs) capable of penetrating intact cancer cells. Once the nanomotor is internalized into the cell, the quenched fluorescence signal (produced by the π–π interaction between GO and a dye-labeled ssDNA) is recovered due to the displacement of the dye-ssDNA probe from the motor GO-quenching surface upon binding with the target miRNA-21, leading to an attractive intracellular “OFF-ON” fluorescence switching. The faster internalization process of the US-powered nanomotors and their rapid movement into the cells increase the likelihood of probe–target contacts, leading to a highly efficient and rapid hybridization. The ability of the nanomotor-based method to screen cancer cells based on the endogenous content of the target miRNA has been demonstrated by measuring the fluorescence signal in two types of cancer cells (MCF-7 and HeLa) with significantly different miRNA-21 expression levels. This single-step, motor-based miRNAs sensing approach enables rapid “on the move” specific detection of the target miRNA-21, even in single cells with an extremely low endogenous miRNA-21 content, allowing precise and real-time monitoring of intracellular miRNA expression