Efficient Delivery of Biological Cargos into Primary Cells by Electrodeposited Nanoneedles via Cell-Cycle-Dependent Endocytosis.

Nanoneedles are a useful tool for delivering exogenous biomolecules to cells. Although therapeutic applications have been explored, the mechanism regarding how cells interact with nanoneedles remains poorly studied. Here, we present a new approach for the generation of nanoneedles, validated their usefulness in cargo delivery, and studied the underlying genetic modulators during delivery. We fabricated arrays of nanoneedles based on electrodeposition and quantified its efficacy of delivery using fluorescently labeled proteins and siRNAs. Notably, we revealed that our nanoneedles caused the disruption of cell membranes, enhanced the expression of cell-cell junction proteins, and downregulated the expression of transcriptional factors of NFκB pathways. This perturbation trapped most of the cells in G2 phase, in which the cells have the highest endocytosis activities. Taken together, this system provides a new model for the study of interactions between cells and high-aspect-ratio materials.

Monitoring Cardiac Biomarkers with Aptamer-Based Molecular Pendulum Sensors.

Reagent-free electronic biosensors capable of analyzing disease markers directly in unprocessed body fluids will enable the development of simple & affordable devices for personalized healthcare monitoring. Here we report a powerful and versatile nucleic acid-based reagent-free electronic sensing system. The signal transduction is based on the kinetics of an electrode-tethered molecular pendulum-a rigid double stranded DNA with one of the strands displaying an analyte-binding aptamer and the other featuring a redox probe-that exhibits field-induced transport modulated by receptor occupancy. Using chronoamperometry, which enables the sensor to circumvent the conventional Debye length limitation, the binding of an analyte can be monitored as these species increase the hydrodynamic drag. The sensing platform demonstrates a low femtomolar quantification limit and minimal cross-reactivity in analyzing cardiac biomarkers in whole blood collected from patients with chronic heart failure.

Capillary-Assisted Molecular Pendulum Bioanalysis.

The development of robust biosensing strategies that can be easily implemented in everyday life remains a challenge for the future of modern biosensor research. While several reagentless approaches have attempted to address this challenge, they often achieve user-friendliness through sacrificing sensitivity or universality. While acceptable for certain applications, these trade-offs hinder the widespread adoption of reagentless biosensing technologies. Here, we report a novel approach to reagentless biosensing that achieves high sensitivity, rapid detection, and universality using the SARS-CoV-2 virus as a model target. Universality is achieved by using nanoscale molecular pendulums, which enables reagentless electrochemical biosensing through a variable antibody recognition element. Enhanced sensitivity and rapid detection are accomplished by incorporating the coffee-ring phenomenon into the sensing scheme, allowing for target preconcentration on a ring-shaped electrode. Using this approach, we obtained limits of detection of 1 fg/mL and 20 copies/mL for the SARS-CoV-2 nucleoproteins and viral particles, respectively. In addition, clinical sample analysis showed excellent agreement with Ct values from PCR-positive SARS-CoV-2 patients.

Strategies for Biomolecular Analysis and Continuous Physiological Monitoring.

Portable devices capable of rapid disease detection and health monitoring are crucial to decentralizing diagnostics from clinical laboratories to the patient point-of-need. Although technologies have been developed targeting this challenge, many require the use of reporter molecules or reagents that complicate the automation and autonomy of sensors. New work in the field has targeted reagentless approaches to enable breakthroughs that will allow personalized monitoring of a wide range of biomarkers on demand. This Perspective focuses on the ability of reagentless platforms to revolutionize the field of sensing by allowing rapid and real-time analysis in resource-poor settings. First, we will highlight advantages of reagentless sensing techniques, specifically electrochemical detection strategies. Advances in this field, including the development of wearable and in situ sensors capable of real-time monitoring of biomarkers such as nucleic acids, proteins, viral particles, bacteria, therapeutic agents, and metabolites, will be discussed. Reagentless platforms which allow for wash-free, calibration free-detection with increased dynamic range are highlighted as a key technological advance for autonomous sensing applications. Furthermore, we will highlight remaining challenges which must be overcome to enable widespread use of reagentless devices. Finally, future prospects and potential breakthroughs in precision medicine that will arise as a result of further development of reagentless sensing approaches are discussed.

Detection of SARS-CoV-2 Viral Particles Using Direct, Reagent-Free Electrochemical Sensing.

The development of new methods for direct viral detection using streamlined and ideally reagent-free assays is a timely and important, but challenging, problem. The challenge of combatting the COVID-19 pandemic has been exacerbated by the lack of rapid and effective methods to identify viral pathogens like SARS-CoV-2 on-demand. Existing gold standard nucleic acid-based approaches require enzymatic amplification to achieve clinically relevant levels of sensitivity and are not typically used outside of a laboratory setting. Here, we report reagent-free viral sensing that directly reads out the presence of viral particles in 5 minutes using only a sensor-modified electrode chip. The approach relies on a class of electrode-tethered sensors bearing an analyte-binding antibody displayed on a negatively charged DNA linker that also features a tethered redox probe. When a positive potential is applied, the sensor is transported to the electrode surface. Using chronoamperometry, the presence of viral particles and proteins can be detected as these species increase the hydrodynamic drag on the sensor. This report is the first virus-detecting assay that uses the kinetic response of a probe/virus complex to analyze the complexation state of the antibody. We demonstrate the performance of this sensing approach as a means to detect, within 5 min, the presence of the SARS-CoV-2 virus and its associated spike protein in test samples and in unprocessed patient saliva.

High-Performance Nucleic Acid Sensors for Liquid Biopsy Applications.

Circulating tumour nucleic acids (ctNAs) are released from tumours cells and can be detected in blood samples, providing a way to track tumors without requiring a tissue sample. This "liquid biopsy" approach has the potential to replace invasive, painful, and costly tissue biopsies in cancer diagnosis and management. However, a very sensitive and specific approach is required to detect relatively low amounts of mutant sequences linked to cancer because they are masked by the high levels of wild-type sequences. This review discusses high-performance nucleic acid biosensors for ctNA analysis in patient samples. We compare sequencing- and amplification-based methods to next-generation sensors for ctDNA and ctRNA (including microRNA) profiling, such as electrochemical methods, surface plasmon resonance, Raman spectroscopy, and microfluidics and dielectrophoresis-based assays. We present an overview of the analytical sensitivity and accuracy of these methods as well as the biological and technical challenges they present.

Three-Dimensional Nanostructured Architectures Enable Efficient Neural Differentiation of Mesenchymal Stem Cells via Mechanotransduction.

Cell morphology and geometry affect cellular processes such as stem cell differentiation, suggesting that these parameters serve as fundamental regulators of biological processes within the cell. Hierarchical architectures featuring micro- and nanotopographical features therefore offer programmable systems for stem cell differentiation. However, a limited number of studies have explored the effects of hierarchical architectures due to the complexity of fabricating systems with rationally tunable micro- and nanostructuring. Here, we report three-dimensional (3D) nanostructured microarchitectures that efficiently regulate the fate of human mesenchymal stem cells (hMSCs). These nanostructured architectures strongly promote cell alignment and efficient neurogenic differentiation where over 85% of hMSCs express microtubule-associated protein 2 (MAP2), a mature neural marker, after 7 days of culture on the nanostructured surface. Remarkably, we found that the surface morphology of nanostructured surface is a key factor that promotes neurogenesis and that highly spiky structures promote more efficient neuronal differentiation. Immunostaining and gene expression profiling revealed significant upregulation of neuronal markers compared to unpatterned surfaces. These findings suggest that the 3D nanostructured microarchitectures can play a critical role in defining stem cell behavior.

Programmable Metal/Semiconductor Nanostructures for mRNA-Modulated Molecular Delivery.

Cytotoxic chemotherapeutics are important tools for the clinical treatment of a variety of solid tumors. However, their use is often complicated by multidrug resistance that can develop in patients, limiting the potencies of these agents. New strategies are needed to provide versatile systems that can respond to and disable resistance mechanisms. We demonstrate the use of a new family of materials, programmable metal/semiconductor nanostructures, for drug delivery and mRNA sensing in drug-resistant cells. These materials are composed of a central core gold nanoparticle surrounded by a layer of DNA-capped quantum dots. The modularity of these "core-satellite" assemblies allows for the construction of superstructures with controlled size and the incorporation of multiple functionalities for drug delivery. The DNA sequence within the nanoparticle specifically binds to an mRNA encoding an important drug resistance factor, MRP1, inside cancer cells, releasing a potent anticancer drug doxorubicin. This event triggers a turn-on fluorescence emission along with a downregulation of the MRP1 drug efflux pump, a main resistance factor for doxorubicin, yielding a remarkable improvement in therapeutic efficacy against drug-resistant cancer cells. This work paves the way for the development of programmable materials with multiple synergistic functionalities for biomedical applications.

Potential-Responsive Surfaces for Manipulation of Cell Adhesion, Release, and Differentiation.

In living systems, interfacial molecular interactions control many biological processes. New stimuli-responsive strategies are desired to provide versatile model systems that can regulate cell behavior in vitro. Described here are potential-responsive surfaces that control cell adhesion and release as well as stem cell differentiation. Cell adhesion can be modulated dynamically by applying negative and positive potentials to surfaces functionalized with tailored monolayers. This process alters cell morphology and ultimately controls behavior and the fate of the cells. Cells can be detached from the electrode surface as intact clusters with different geometries using electrochemical potentials. Importantly, morphological changes during adhesion guide stem cell differentiation. The higher accessibility of the peptide under a positive applied potential causes phenotypic changes in the cells that are hallmarks of osteogenesis, whereas lower accessibility of the peptide promoted by negative potentials leads to adipogenesis.

High-Curvature Nanostructuring Enhances Probe Display for Biomolecular Detection.

High-curvature electrodes facilitate rapid and sensitive detection of a broad class of molecular analytes. These sensors have reached detection limits not attained using bulk macroscale materials. It has been proposed that immobilized DNA probes are displayed at a high deflection angle on the sensor surface, which allows greater accessibility and more efficient hybridization. Here we report the first use of all-atom molecular dynamics simulations coupled with electrochemical experiments to explore the dynamics of single-stranded DNA immobilized on high-curvature versus flat surfaces. We find that high-curvature structures suppress DNA probe aggregation among adjacent probes. This results in conformations that are more freely accessed by target molecules. The effect observed is amplified in the presence of highly charged cations commonly used in electrochemical biosensing. The results of the simulations agree with experiments that measure the degree of hybridization in the presence of mono-, di-, and trivalent cations. On high-curvature structures, hybridization current density increases as positive charge increases, whereas on flat electrodes, the trivalent cations cause aggregation due to electrostatic overscreening, which leads to decreased current density and less sensitive detection.

Combinatorial Probes for High-Throughput Electrochemical Analysis of Circulating Nucleic Acids in Clinical Samples.

The analysis of circulating tumour nucleic acids (ctNAs) provides a minimally invasive way to assess the mutational spectrum of a tumour. However, effective and practical methods for analyzing this emerging class of markers are lacking. Analysis of ctNAs using a sensor-based approach has notable challenges, as it is vital to differentiate nucleic acids from normal cells from mutation-bearing sequences emerging from tumours. Moreover, many genes related to cancer have dozens of different mutations. Herein, we report an electrochemical approach that directly detects genes with mutations in patient serum by using combinatorial probes (CPs). The CPs enable detection of all of the mutant alleles derived from the same part of the gene. As a proof of concept, we analyze mutations of the EGFR gene, which has more than 40 clinically relevant alterations that include deletions, insertions, and point mutations. Our CP-based approach accurately detects mutant sequences directly in patient serum.

DNA Clutch Probes for Circulating Tumor DNA Analysis.

Progress toward the development of minimally invasive liquid biopsies of disease is being bolstered by breakthroughs in the analysis of circulating tumor DNA (ctDNA): DNA released from cancer cells into the bloodstream. However, robust, sensitive, and specific methods of detecting this emerging analyte are lacking. ctDNA analysis has unique challenges, since it is imperative to distinguish circulating DNA from normal cells vs mutation-bearing sequences originating from tumors. Here we report the electrochemical detection of mutated ctDNA in samples collected from cancer patients. By developing a strategy relying on the use of DNA clutch probes (DCPs) that render specific sequences of ctDNA accessible, we were able to readout the presence of mutated ctDNA. DCPs prevent reassociation of denatured DNA strands: they make one of the two strands of a dsDNA accessible for hybridization to a probe, and they also deactivate other closely related sequences in solution. DCPs ensure thereby that only mutated sequences associate with chip-based sensors detecting hybridization events. The assay exhibits excellent sensitivity and specificity in the detection of mutated ctDNA: it detects 1 fg/µL of a target mutation in the presence of 100 pg/µL of wild-type DNA, corresponding to detecting mutations at a level of 0.01% relative to wild type. This approach allows accurate analysis of samples collected from lung cancer and melanoma patients. This work represents the first detection of ctDNA without enzymatic amplification.

Arsenic(III) detection using electrochemical-chemical-chemical redox cycling at bare indium-tin oxide electrodes.

Sensitive As(III) detection in ground water is of great importance for evaluating the quality of drinking water. We report a sensitive electrochemical method for As(III) detection based on electrochemical-chemical-chemical (ECC) redox cycling involving Ru(IV) [an oxidized species of Ru(III)(NH3)5NH2(2+)], As(III), and tris(3-carboxyethyl)phosphine (TCEP). Electrochemical oxidation of Ru(III)(NH3)5NH2(2+) formed from Ru(III)(NH3)6(3+) generates Ru(IV), which quickly oxidizes As(III). This electro-mediated oxidation of As(III) produces As(V), which is reduced back to As(III) by TCEP. Electrochemically generated Ru(IV) then reoxidizes As(III), allowing ECC redox cycling to occur at a high rate on bare indium-tin oxide (ITO) electrodes without modifying the surfaces with electrocatalytic materials. Because most interfering metal ions precipitate in a carbonate buffer, water samples are mixed with carbonate buffers prior to electrochemical measurements, rendering the effects of Cu(+), Cu(2+), Fe(2+), Fe(3+), and Pb(2+) insignificant. The detection limit calculated by ECC redox cycling using a chronocoulogram is 1.2 µM, much lower than that obtained using only the electro-mediated oxidation of As(III) (90 µM).

Tuning the bacterial detection sensitivity of nanostructured microelectrodes.

Fast, sensitive nucleic acid sensors that enable direct detection of bacteria and diagnosis of infectious disease would offer significant advantages over existing approaches that employ enzymatic amplification of nucleic acids. We have developed chip-based microelectrodes that are highly effective for bacterial detection and have shown that they can capture and permit the analysis of large slow moving mRNA targets. Here, we explore new approaches to tune their analytical sensitivity and investigate the effect of sensor size, material composition, and probe density on the electrochemical signals obtained in the presence of bacteria. Sensor size can be varied from 10 to 100 µm, and this parameter can change detection limits obtained by a factor of 100. Changing the surface coating can also be used to tune sensitivity, with more nanostructured coatings yielding the most sensitive detectors. Moreover, we assessed performance of the sensors by tuning probe density. Varying the density of the immobilized probe had a dramatic effect on sensitivity, with sparse probe monolayers providing superior levels of performance. Overall, this study points to several factors that can be used to tune detection limits.

Wearable nucleic acid testing platform - A perspective on rapid self-diagnosis and surveillance of infectious diseases.

Wearable biosensors (WB) are currently attracting considerable interest for rapid detection and monitoring of biomarkers including metabolites, protein, and pathogen in bodily fluids (e.g., sweat, saliva, tears, and interstitial fluid). Another branch of WB termed wearable nucleic acid testing (NAT) is blossoming thanks to the development of microfluidic technology and isothermal nucleic acid amplification technique (iNAAT); however, there are only few reports on this. The wearable NAT is an emerging field of point-of-care (POC) diagnostics, and holds the promise for time-saving self-diagnosis, and evidence-based surveillance of infectious diseases in remote or low-resource settings. The use of wearable NAT can also be advanced to include molecular diagnosis, the identification of cancer biomarkers, genetic abnormalities, and other aspects. The wearable NAT provides the potential for evidence-based surveillance of infectious diseases when combined with internet connectivity and App software. To make the wearable NAT accessible to the end users, however, improvements must be made to the fabrication, cost, speed, sensitivity, specificity, sampling, iNAAT, analyzer, and a few other features. So, in this paper, we looked at the wearable NAT's most recent development, identified its difficulties, and defined its potential for managing infectious diseases quickly in the future. This is the wearable NAT review's first effort. We expect that this article will provide the concise resources needed to develop and deploy an efficient wearable NAT system.

Biomolecular sensors for advanced physiological monitoring.

Body-based biomolecular sensing systems, including wearable, implantable and consumable sensors allow comprehensive health-related monitoring. Glucose sensors have long dominated wearable bioanalysis applications owing to their robust continuous detection of glucose, which has not yet been achieved for other biomarkers. However, access to diverse biological fluids and the development of reagentless sensing approaches may enable the design of body-based sensing systems for various analytes. Importantly, enhancing the selectivity and sensitivity of biomolecular sensors is essential for biomarker detection in complex physiological conditions. In this Review, we discuss approaches for the signal amplification of biomolecular sensors, including techniques to overcome Debye and mass transport limitations, and selectivity improvement, such as the integration of artificial affinity recognition elements. We highlight reagentless sensing approaches that can enable sequential real-time measurements, for example, the implementation of thin-film transistors in wearable devices. In addition to sensor construction, careful consideration of physical, psychological and security concerns related to body-based sensor integration is required to ensure that the transition from the laboratory to the human body is as seamless as possible.

A magneto-activated nanoscale cytometry platform for molecular profiling of small extracellular vesicles.

Exosomal PD-L1 (exoPD-L1) has recently received significant attention as a biomarker predicting immunotherapeutic responses involving the PD1/PD-L1 pathway. However, current technologies for exosomal analysis rely primarily on bulk measurements that do not consider the heterogeneity found within exosomal subpopulations. Here, we present a nanoscale cytometry platform NanoEPIC, enabling phenotypic sorting and exoPD-L1 profiling from blood plasma. We highlight the efficacy of NanoEPIC in monitoring anti-PD-1 immunotherapy through the interrogation of exoPD-L1. NanoEPIC generates signature exoPD-L1 patterns in responders and non-responders. In mice treated with PD1-targeted immunotherapy, exoPD-L1 is correlated with tumor growth, PD-L1 burden in tumors, and the immune suppression of CD8+ tumor-infiltrating lymphocytes. Small extracellular vesicles (sEVs) with different PD-L1 expression levels display distinctive inhibitory effects on CD8 + T cells. NanoEPIC offers robust, high-throughput profiling of exosomal markers, enabling sEV subpopulation analysis. This platform holds the potential for enhanced cancer screening, personalized treatment, and therapeutic response monitoring.

Protein detection using arrayed microsensor chips: tuning sensor footprint to achieve ultrasensitive readout of CA-125 in serum and whole blood.

Multiplexed assays that can measure protein biomarkers and internal standards are highly desirable given the potential to reduce false positives and negatives. We report here the use of a chip-based platform that achieves multiplexed immunosensing of the ovarian cancer biomarker CA-125 without the need for covalent labeling or sandwich complexes. The sensor chips allow the straightforward comparison of detectors of different sizes, and we used this feature to scan the microscale size regime for the best sensor size and optimize the limit of detection exhibited down to 0.1 U/mL. The assay has a straightforward design, with readout being performed in a single step involving the introduction of a noncovalently attached redox reporter group. The detection system reported exhibits excellent specificity, with analysis of a specific cancer biomarker, CA-125, performed in human serum and whole blood. The multiplexing of the system allows the analysis of the biomarker to be performed in parallel with an abundant serum protein for internal calibration.

Reagentless biomolecular analysis using a molecular pendulum.

The development of reagentless sensors that can detect molecular analytes in biological fluids could enable a broad range of applications in personalized health monitoring. However, only a limited set of molecular inputs can currently be detected using reagentless sensors. Here, we report a sensing mechanism that is compatible with the analysis of proteins that are important physiological markers of stress, allergy, cardiovascular health, inflammation and cancer. The sensing method is based on the motion of an inverted molecular pendulum that exhibits field-induced transport modulated by the presence of a bound analyte. We measure the sensor's electric field-mediated transport using the electron-transfer kinetics of an attached reporter molecule. Using time-resolved electrochemical measurements that enable unidirectional motion of our sensor, the presence of an analyte bound to our sensor complex can be tracked continuously in real time. We show that this sensing approach is compatible with making measurements in blood, saliva, urine, tears and sweat and that the sensors can collect data in situ in living animals.

Ultrasensitive detection of DNA in diluted serum using NaBH4 electrooxidation mediated by [Ru(NH3)6]3+ at indium-tin oxide electrodes.

There is a crucial need for simple and highly sensitive techniques to detect DNA in complicated biological samples such as serum. Here we present an ultrasensitive electrochemical DNA sensor using (i) single DNA hybridization with peptide nucleic acid (PNA), (ii) selective binding of [Ru(NH(3))(6)](3+) to hybridized DNA, (iii) fast NaBH(4) electrooxidation mediated by [Ru(NH(3))(6)](3+), and (iv) low background currents of NaBH(4) at indium-tin oxide (ITO) electrodes. The [Ru(III)(NH(3))(5)NH(2)](2+) formed from [Ru(III)(NH(3))(6)](3+) in borate buffer (pH 11.0) is readily electrooxidized to both [Ru(IV)(NH(3))(5)NH(2)](3+) and Ru complex with a higher oxidation state. In the absence of [Ru(NH(3))(6)](3+) bound to the DNA-sensing ITO electrodes, the oxidation currents of NaBH(4) are very low. However, in the presence of [Ru(NH(3))(6)](3+), the oxidation currents of NaBH(4) are highly enhanced due to electron mediation of the oxidized Ru complexes. The significant enhancement in the electrocatalytic activity of sensing electrodes after [Ru(NH(3))(6)](3+) binding facilitates to obtain high signal-to-background ratios. PNA and ethylenediamine on DNA-sensing electrodes significantly decrease [Ru(NH(3))(6)](3+) binding, also allowing for high signal-to-background ratios. The oxidation charges of NaBH(4) obtained from chronocoulometry are highly reproducible. All combined effects enable the detection of DNA with a detection limit of 1 fM in ten-fold diluted human serum. The simple and fast detection procedure and the ultrasensitivity make this approach highly promising for practical DNA detection.