Ultrasensitive Detection of Dopamine, IL-6 and SARS-CoV-2 Proteins on Crumpled Graphene FET Biosensor.

Universal platforms for biomolecular analysis using label-free sensing modalities can address important diagnostic challenges. Electrical field effect-sensors are an important class of devices that can enable point-of-care sensing by probing the charge in the biological entities. Use of crumpled graphene for this application is especially promising. It is previously reported that the limit of detection (LoD) on electrical field effect-based sensors using DNA molecules on the crumpled graphene FET (field-effect transistor) platform. Here, the crumpled graphene FET-based biosensing of important biomarkers including small molecules and proteins is reported. The performance of devices is systematically evaluated and optimized by studying the effect of the crumpling ratio on electrical double layer (EDL) formation and bandgap opening on the graphene. It is also shown that a small and electroneutral molecule dopamine can be captured by an aptamer and its conformation change induced electrical signal changes. Three kinds of proteins were captured with specific antibodies including interleukin-6 (IL-6) and two viral proteins. All tested biomarkers are detectable with the highest sensitivity reported on the electrical platform. Significantly, two COVID-19 related proteins, nucleocapsid (N-) and spike (S-) proteins antigens are successfully detected with extremely low LoDs. This electrical antigen tests can contribute to the challenge of rapid, point-of-care diagnostics.

Detection of SARS-CoV-2 Virus Amplification Using a Crumpled Graphene Field-Effect Transistor Biosensor.

The rapid and unexpected spread of SARS-CoV-2 worldwide has caused unprecedented disruption to daily life and has brought forward critical challenges for public health. The disease was the largest cause of death in the United States in early 2021. Likewise, the COVID-19 pandemic has highlighted the need for rapid and accurate diagnoses at scales larger than ever before. To improve the availability of current gold standard diagnostic testing methods, the development of point-of-care devices that can maintain gold standard sensitivity while reducing the cost and providing portability is much needed. In this work, we combine the amplification capabilities of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) techniques with high-sensitivity end-point detection of crumpled graphene field-effect transistors (cgFETs) to develop a portable detection cell. This electrical detection method takes advantage of the ability of graphene to adsorb single-stranded DNA due to noncovalent π-π bonds but not double-stranded DNA. These devices have demonstrated the ability to detect the presence of the SARS-CoV-2 virus in a range from 10 to 104 copies/µL in 20 viral transport medium (VTM) clinical samples. As a result, we achieved 100% PPV, NPV, sensitivity, and specificity with 10 positive and 10 negative VTM clinical samples. Further, the cgFET devices can differentiate between positive and negative VTM clinical samples in 35 min based on the Dirac point shift. Likewise, the improved sensing capabilities of the crumpled gFET were compared with those of the traditional flat gFET devices.

Ultrasensitive detection of nucleic acids using deformed graphene channel field effect biosensors.

Field-effect transistor (FET)-based biosensors allow label-free detection of biomolecules by measuring their intrinsic charges. The detection limit of these sensors is determined by the Debye screening of the charges from counter ions in solutions. Here, we use FETs with a deformed monolayer graphene channel for the detection of nucleic acids. These devices with even millimeter scale channels show an ultra-high sensitivity detection in buffer and human serum sample down to 600 zM and 20 aM, respectively, which are ∼18 and ∼600 nucleic acid molecules. Computational simulations reveal that the nanoscale deformations can form 'electrical hot spots' in the sensing channel which reduce the charge screening at the concave regions. Moreover, the deformed graphene could exhibit a band-gap, allowing an exponential change in the source-drain current from small numbers of charges. Collectively, these phenomena allow for ultrasensitive electronic biomolecular detection in millimeter scale structures.

Nucleus-Targeted, Echogenic Polymersomes for Delivering a Cancer Stemness Inhibitor to Pancreatic Cancer Cells.

Chemotherapeutic agents for treating cancers show considerable side effects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport and subsequently release the encapsulated anticancer drugs within the nuclei of pancreatic cancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3-polyethylene glycol (PEG)-polylactic acid (PLA) copolymer employing the Cu2+ catalyzed "Click" reaction. We prepared polymersomes from the dexamethasone-PEG-PLA conjugate along with a synthesized stimuli-responsive polymer PEG-S-S-PLA. The dexamethasone group dilates the nuclear pore complexes and transports the vesicles to the nuclei. We designed the polymersomes to release the encapsulated drugs in the presence of a high concentration of reducing agents in the nuclei of pancreatic cancer cells. We observed that the nucleus-targeted, stimuli-responsive polymersomes released 70% of encapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulated the cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608 encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. The polymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medical ultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have the potential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Quantitative measurements of dielectrophoresis in a nanoscale electrode array with an atomic force microscopy.

Nanoelectronic devices integrated with dielectrophoresis (DEP) have been promoted as promising platforms for trapping, separating, and concentrating target biomarkers and cancer cells from a complex medium. Here, we visualized DEP and DEP gradients in conventional nanoelectronic devices by using multi-pass atomic force microcopy techniques. Our measurements directly demonstrated a short range DEP only at sharp step edges of electrodes, frequency dependent DEP polarity, and separation distance dependent DEP strength. Additionally, non-uniform DEP along the edges of the electrodes due to a large variation in electric field strength was observed. The strength and apparent working distance of DEP were measured to be an order of a few nN and 80 nm within the limited scale of particles and other parameters such as an ionic strength of the medium. This method provides a powerful tool to quantify the strength and polarity of DEP and allows optimizing and calibrating the device's operating parameters including the driving field strength for the effective control and manipulation of target biomolecules.

Nonmonotonic DNA-length-dependent mobility in pluronic gels.

Two-dimensional electrophoresis was used to analyze the mobility of DNA fragments in micellar gels of pluronic F127 (EO_{100}PO_{70}EO_{100}) and pluronic P123 (EO_{20}PO_{70}EO_{20}). The 20-3500 base pair DNA fragments were separated by size first in agarose gels, and then in pluronic gels at room temperature. In agarose gels, the DNA mobility decreases monotonically with increasing DNA length. In pluronic gels, however, the mobility varies nonmonotonically according to fragment lengths that are strongly correlated with the diameter of the spherical micelles. Brownian dynamics (BD) simulations with short-ranged intra-DNA hydrodynamic interactions were performed to numerically calculate the length-dependent mobility in pluronic lattices. The rising and falling trends, as well as the oscillations of mobility, were captured by the coarse-grained BD simulations. Molecular dynamics simulations in pluronic F127, with explicitly modeled micelle coronas, justified that the hydrodynamic interactions mediated by the complex fluid of hydrated poly(ethylene oxide) are a possible reason for the initial rise of mobility with DNA length.

Real-time monitoring of conformational transitions of single-molecule histone deacetylase 8 with nanocircuits.

Using single-molecule approaches, we directly observed the dynamic interaction between HDAC8 and various ligands as well as conformational interconversions during the catalytic reaction. Statistical analysis identified key kinetic parameters, demonstrating that the enzymatic activity is highly sensitive to both minor variations in the ligand structures and small synthetic molecules.

Prostate-Specific Membrane Antigen Targeted Polymersomes for Delivering Mocetinostat and Docetaxel to Prostate Cancer Cell Spheroids.

Prostate cancer cells overexpress the prostate-specific membrane antigen (PSMA) receptors on the surface. Targeting the PSMA receptor creates a unique opportunity for drug delivery. Docetaxel is a Food and Drug Administration-approved drug for treating metastatic and androgen-independent prostate cancer, and mocetinostat is a potent inhibitor of class I histone deacetylases. In this study, we prepared reduction-sensitive polymersomes presenting folic acid on the surface and encapsulating either docetaxel or mocetinostat. The presence of folic acid allowed efficient targeting of the PSMA receptor and subsequent internalization of the polymeric vesicles in cultured LNCaP prostate cancer cell spheroids. The intracellular reducing agents efficiently released docetaxel and mocetinostat from the polymersomes. The combination of the two drug-encapsulated polymersome formulations significantly (p < 0.05) decreased the viability of the LNCaP cells (compared to free drugs or control) in three-dimensional spheroid cultures. The calculated combination index value indicated a synergistic effect for the combination of mocetinostat and docetaxel. Thus, our PSMA-targeted drug-encapsulated polymersomes has the potential to lead to a new direction in prostate cancer therapy that decreases the toxicity and increases the efficacy of the drug delivery systems.

Hypoxia Responsive, Tumor Penetrating Lipid Nanoparticles for Delivery of Chemotherapeutics to Pancreatic Cancer Cell Spheroids.

Solid tumors are often poorly irrigated due to structurally compromised microcirculation. Uncontrolled multiplication of cancer cells, insufficient blood flow, and the lack of enough oxygen and nutrients lead to the development of hypoxic regions in the tumor tissues. As the partial pressure of oxygen drops below the necessary level (10 psi), the cancer cells modulate their genetic makeup to survive. Hypoxia triggers tumor progression by enhancing angiogenesis, cancer stem cell production, remodeling of the extracellular matrix, and epigenetic changes in the cancer cells. However, the hypoxic regions are usually located deep in the tumors and are usually inaccessible to the intravenously injected drug carrier or the drug. Considering the designs of the reported nanoparticles, it is likely that the drug is delivered to the peripheral tumor tissues, close to the blood vessels. In this study, we prepared lipid nanoparticles (LNs) comprising the synthesized hypoxia-responsive lipid and a peptide-lipid conjugate. We observed that the resultant LNs penetrated to the hypoxic regions of the tumors. Under low oxygen partial pressure, the hypoxia-responsive lipid undergoes reduction, destabilizing the lipid membrane, and releasing encapsulated drugs from the nanoparticles. We demonstrated the results employing spheroidal cultures of the pancreatic cancer cells BxPC-3. We observed that the peptide-decorated, drug encapsulated LNs reduced the viability of pancreatic cancer cells of the spheroids to 35% under hypoxic conditions.

Hypoxia-Responsive Polymersomes for Drug Delivery to Hypoxic Pancreatic Cancer Cells.

Hypoxia in tumors contributes to overall tumor progression by assisting in epithelial-to-mesenchymal transition, angiogenesis, and metastasis of cancer. In this study, we have synthesized a hypoxia-responsive, diblock copolymer poly(lactic acid)-azobenzene-poly(ethylene glycol), which self-assembles to form polymersomes in an aqueous medium. The polymersomes did not release any encapsulated contents for 50 min under normoxic conditions. However, under hypoxia, 90% of the encapsulated dye was released in 50 min. The polymersomes encapsulated the combination of anticancer drugs gemcitabine and erlotinib with entrapment efficiency of 40% and 28%, respectively. We used three-dimensional spheroid cultures of pancreatic cancer cells BxPC-3 to demonstrate hypoxia-mediated release of the drugs from the polymersomes. The vesicles were nontoxic. However, a significant decrease in cell viability was observed in hypoxic spheroidal cultures of BxPC-3 cells in the presence of drug encapsulated polymersomes. These polymersomes have potential for future applications in imaging and treatment of hypoxic tumors.

Acridine Orange Conjugated Polymersomes for Simultaneous Nuclear Delivery of Gemcitabine and Doxorubicin to Pancreatic Cancer Cells.

Considering the systemic toxicity of chemotherapeutic agents, there is an urgent need to develop new targeted drug delivery systems. Herein, we have developed a new nuclear targeted, redox sensitive, drug delivery vehicle to simultaneously deliver the anticancer drugs gemcitabine and doxorubicin to the nuclei of pancreatic cancer cells. We prepared polymeric bilayer vesicles (polymersomes), and actively encapsulated the drug combination by the pH gradient method. A redox-sensitive polymer (PEG-S-S-PLA) was incorporated to sensitize the formulation to reducing agent concentration. Acridine orange (AO) was conjugated to the surface of the polymersomes imparting nuclear localizing property. The polymersomes' toxicity and efficacy were compared with those of a free drug combination using monolayer and three-dimensional spheroid cultures of pancreatic cancer cells. We observed that the redox sensitive, nuclear-targeted polymersomes released more than 60% of their encapsulated contents in response to 50 mM glutathione. The nanoparticles are nontoxic; however, the drug encapsulated vesicles have significant toxicity. The prepared formulation can increase the drug's therapeutic index by delivering the drugs directly to the cells' nuclei, one of the key organelles in the cells. This study is likely to initiate research in targeted nuclear delivery using other drug formulations in other types of cancers.

Single molecule observation of DNA electrophoresis in pluronic f127.

Single molecule fluorescence microscopy is used to follow the motion of long DNA molecules undergoing electrophoresis in Pluronic gels. We find that for low fields most DNA molecules follow tortuous paths through the gels, at an angle up to 90 degrees from the field direction, while some molecules find paths along the field lines. In high fields, virtually all of the DNA molecules follow the field lines. In many cases, the molecules travel as compact coils with optically discernible radii smaller than in free solution. In other cases, the molecules extend and contract or travel in an extended configuration.