Recent advances in enzymatic biosensors for point-of-care detection of biomolecules.

Late state-of-the-art analytical methodologies in chromatography, spectroscopy, and electroanalysis have been developed to meet the challenges of changing environmental and health issues. The modern trends in developing new protocols emphasize economic, portable, nano, or even smaller sample sizes and less time-consuming processes. This has led to the development of technology-based biosensors which meet most of the above requirements. The lab-on-chip technology exploiting enzyme-based biosensors has made the analytical processes very efficient, accurate, affordable, and requiring nano-scale sample sizes. In this review, an attempt is being made to review the literature based on state-of-the-art technology of enzyme-based biosensors for the detection of biomolecules.

Efficient Point-of-Care Detection of Uric Acid in the Human Blood Sample with an Enhanced Electrocatalytic Response Using Nanocomposites of Cobalt and Mixed-Valent Molybdenum Sulfide.

This work efficiently detects uric acid (UA) in a human blood sample using cobalt nanoparticle-immobilized mixed-valent molybdenum sulfide on the copper substrate in a point-of-care (PoC) device. The sensor electrode was fabricated by micromachining of Cu clad boards employing an engraver to generate a three-electrode system consisting of working electrode (WE), reference electrode (RE), and counter electrode (CE). The WE was subjected to physical vapor deposition of mixed-valent MoSx layers by a reaction between Mo(CO)6 and H2S at ∼200 °C using a simple setup following which CoNPs were electrochemically deposited. The RE and CE were covered with Ag/AgCl and Ag paste, respectively. A plasma separation membrane acted as the medium of UA/blood serum delivery to the electrodes. The material and electrochemical characterization confirmed that CoNPs over MoSx provided an enlarged electroactive surface for the direct electron transfer to achieve an enhanced electrocatalytic response. The binary combination of CoNPs and MoSx layers over the Cu electrode reduced the charge-transfer resistance by two times, enhanced the surface adsorption by more than two times, and yielded a high diffusion coefficient of 3.46 × 10-3 cm2/s. These interfacial effects facilitated the UA oxidation, leading to unprecedented mA range current density for UA sensing for the PoC device. The electrochemical detection tests in the PoC device revealed a sensitivity of 64.7 µA/µM cm-2, which is ∼50 times higher compared to the latest reported value (1.23 µA/µM cm-2), a high limit of detection of 5 nM, and shelf life of 6 months, confirming the synergistic effect-mediated high sensitivity under PoC settings. Interference tests confirmed no intervention of similar analytes. Tests on blood samples demonstrated a recovery percentage close to 100% in human serum UA, signifying the suitability of the nanocomposite-based sensor and the PoC device for clinical sensing applications.

Bi-Functional Gold Nanorod-Protein Conjugates with Biomimetic BSA@Folic Acid Corona for Improved Tumor Targeting and Intracellular Delivery of Therapeutic Proteins in Colon Cancer 3D Spheroids.

Gold nanorods (AuNRs) remain well-developed inorganic nanocarriers of small molecules for a plethora of biomedical and therapeutic applications. However, the delivery of therapeutic proteins using AuNRs with high protein loading capacity (LC), serum stability, excellent target specificity, and minimal off-target protein release is not known. Herein, we report two bi-functional AuNR-protein nanoconjugates, AuNR@EGFP-BSAFA and AuNR@RNaseA-BSAFA, supramolecularly coated with folic acid-modified BSA (BSAFA) acting as biomimetic protein corona to demonstrate targeted cytosolic delivery of enhanced green fluorescent protein (EGFP) and therapeutic ribonuclease A enzyme (RNase A) in their functional forms. AuNR@EGFP-BSAFA and AuNR@RNaseA-BSAFA exhibit high LCs of ∼42 and ∼54%, respectively, increased colloidal stability, and rapid protein release in the presence of biological thiols. As a nanocarrier, AuNR@EGFP-BSAFA and AuNR@RNaseA-BSAFA show resistance to corona formation in high-serum media even after 24 h, guaranteeing a greater circulation lifetime. Folate receptor-targeting BSAFA on the AuNR surface facilitates the receptor-mediated internalization, followed by the release of EGFP and RNase A in HT29 cells. The green fluorescence dispersed throughout the cell's cytoplasm indicates successful cytosolic delivery of EGFP by AuNR@EGFP-BSAFA. AuNR@RNaseA-BSAFA-mediated therapeutic RNase A delivery in multicellular 3D spheroids of HT29 cells exhibits a radical reduction in the cellular RNA fluorescence intensity to 38%, signifying RNA degradation and subsequent cell death. The versatile nanoformulation strategy in terms of the anisotropic particle morphology, protein type, and ability for targeted delivery in the functional form makes the present AuNR-protein nanoconjugates a promising platform for potential application in cancer management.

Advances in Thiopurine Drug Delivery: The Current State-of-the-Art.

Thiopurines (mercaptopurine, azathioprine and thioguanine) are well-established maintenance treatments for a wide range of diseases such as leukemia, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE) and other inflammatory and autoimmune diseases in general. Worldwide, millions of patients are treated with thiopurines. The use of thiopurines has been limited because of off-target effects such as myelotoxicity and hepatotoxicity. Therefore, seeking methods to enhance target-based thiopurine-based treatment is relevant, combined with pharmacogenetic testing. Controlled-release formulations for thiopurines have been clinically tested and have shown promising outcomes in inflammatory bowel disease. Latest developments in nano-formulations for thiopurines have shown encouraging pre-clinical results, but further research and development are needed. This review provides an overview of novel drug delivery strategies for thiopurines, reviewing modified release formulations and with a focus on nano-based formulations.

3-D printed electrode integrated sensing chip and a PoC device for enzyme free electrochemical detection of blood urea.

Herein we report a novel electrochemical sensing chip and a point-of-care device (PoC) for enzyme-free electrochemical detection of urea in human blood. The electrochemical sensing chip was developed by 3-D printing of conductive Ag ink and subsequent electrodeposition of AuNP-rGO nanocomposite. Material characterization of the sensing chip was conducted to find a plausible mechanism for the electrochemical reaction with urea. Subsequently, the response with varying concentrations of urea in solution and human blood samples was tested. High peak response current (~5 times than that of the highest reported value), low impedance, rapid sensor fabrication procedure, high selectivity towards urea, excellent linear response (R2 = 0.99), high sensitivity of 183 µA mM-1 cm-2, the fast response indicated by high diffusion coefficient, the limit of detection of 0.1 µM, tested shelf life of more than 6 months and recovery rate of >99% ensured the application of the developed sensor chip towards PoC urea detection test kit. A PoC device housing an electronic circuitry following the principles of linear sweep voltammetry and compatible with a sensing chip was developed. A maximum percentage error of 4.86% and maximum RSD of 3.63% confirmed the use of the PoC device for rapid urea measurements in human blood.

Facile Detection of Blood Creatinine Using Binary Copper-Iron Oxide and rGO-Based Nanocomposite on 3D Printed Ag-Electrode under POC Settings.

Metal nanoparticles have been helpful in creatinine sensing technology under point-of-care (POC) settings because of their excellent electrocatalyst properties. However, the behavior of monometallic nanoparticles as electrochemical creatinine sensors showed limitations concerning the current density in the mA/cm2 range and wide detection window, which are essential parameters for the development of a sensor for POC applications. Herein, we report a new sensor, a reduced graphene oxide stabilized binary copper-iron oxide-based nanocomposite on a 3D printed Ag-electrode (Fe-Cu-rGO@Ag) for detecting a wide range of blood creatinine (0.01 to 1000 µM; detection limit 10 nM) in an electrochemical chip with a current density ranging between 0.185 and 1.371 mA/cm2 and sensitivity limit of 1.1 µA µM-1 cm-2 at physiological pH. Interference studies confirmed that the sensor exhibited no interference from analytes like uric acid, urea, dopamine, and glutathione. The sensor response was also evaluated to detect creatinine in human blood samples with high accuracy in less than a minute. The sensing mechanism suggested that the synergistic effects of Cu and iron oxide nanoparticles played an essential role in the efficient sensing where Fe atoms act as active sites for creatinine oxidation through the secondary amine nitrogen, and Cu nanoparticles acted as an excellent electron-transfer mediator through rGO. The rapid sensor fabrication procedure, mA/cm2 peak current density, a wide range of detection limits, low contact resistance including high selectivity, excellent linear response (R2 = 0.991), and reusability ensured the application of advanced electrochemical sensor toward the POC creatinine detection.

Printed oxygen gas sensor using copper-DTDTPA solid electrolyte.

We present a new method for the rapid and cost-effective fabrication of solid electrolyte-based printed potentiometric oxygen sensors working at ambient temperature using Cu-dithiolated diethylene triamine pentaacetic acid complex molecules (Cu-DTDTPA) adsorbed on Grade-1 laboratory filter paper and subsequent 3-D printing of interdigitated electrodes employing silver/silver chloride ink. The decrease in conductivity with time and frequency-dependent impedance response confirms the filter paper adsorbed Cu-DTDTPA as a solid electrolyte. A plausible structure of the Cu-DTDTPA solid electrolyte and its mechanism of reaction with oxygen are presented. A maximum sensitivity of 0.052 mV per %O2, the maximum response time of 1.15 s per %O2, a wide measurement output ranging from 14.55 mV to 17.25 mV for 20%-90% of O2 concentration, a maximum standard deviation of 0.12 mV in output voltage, almost similar trends of the response on temperature, humidity variations and ageing and high selectivity establish the sensor for use in medical ventilator applications, specifically in the COVID19 pandemic.

Development of 6-Thioguanine conjugated PLGA nanoparticles through thioester bond formation: Benefits of electrospray mediated drug encapsulation and sustained release in cancer therapeutic applications.

Polymeric nanoparticle-based successful delivery of hydrophobic drugs is highly desirable for its controlled and sustained release at the disease site, which is a challenge with the current synthesis methods. In the present study, an electrospray mediated facile one-step synthesis approach is explored in which a solution mixture of a hydrophobic drug, 6-thioguanine (Tg) and a biocompatible FDA approved polymer, Poly (d, l-lactide-co-glycolide) (PLGA) is injected in an applied electric field of suitable intensity to prepare drug encapsulated PLGA nanoparticles, PLGA-Tg with high yield. In order to explore the effect of external electric field on Tg loading and delivery applications, the nanoparticles are characterized using EDX, AFM, FESEM, TEM, FTIR, Raman, fluorescence, and mass spectroscopy techniques. The characterization studies indicate that the electric field mediated synthesis exhibits spherical nanoparticles with a homogenous core size distribution of ~60 nm, high encapsulation (~97.22%) and stable conjugation of Tg (via thioester linkages) with PLGA molecules in the presence of the applied electric field. The kinetic study demonstrates the 'anomalous diffusion' (non-Fickian diffusion) release mechanism in which Tg escapes from PLGA matrix with a slow, but steady diffusion rate and the sustained drug release profile continues for 60 days. To check the biological activity of the encapsulated Tg, in-vitro cell studies of the PLGA-Tg are performed on HeLa cells. The MTT assay shows significant cell death after 48 h of treatment, and the cellular internalization of the drug-loaded nanoparticles occurs through pinocytosis mediated uptake, which is established by the AFM analysis. The Raman and mass spectroscopy studies suggest that the PLGA-Tg nanoparticles are rapidly hydrolyzed inside cell cytoplasm to release Tg which initiates apoptosis-mediated cell death confirmed by as DNA fragmentation and membrane blebbing studies. The results clearly emphasize the benefits of electrospray based synthesis of polymeric nanodrug formulation through the formation of chemical bonds between polymer and drug molecules that could be easily implemented in the design and development of an effective nanotherapeutic platform with no typical 'burst effect,' prolonged release profile, and significant toxicity to the cancer cells.

Gold-nanoparticle-embedded microchannel array for enhanced power generation.

A high streaming potential and current were generated using a gold-nanoparticle-embedded patterned PDMS microchannel array. Gold nanoparticles with dimensions of ∼70 nm were prepared inside a hydrophobic patterned PDMS microchannel. The channel array was developed on a ridge-shaped patterned surface by performing soft lithography using UV-laser micromachining with a ridge spacing of 27.0 µm, width of 22.0 µm, and height of 16.0 µm. Subsequently, tests were conducted in which ultrapure water, solutions of 0.1 M NaCl, 0.1 M HCl and 40% H2O2 were passed through the patterned channel array at various flow rates and pressures using a microfluidic pump wherein the channel inlet and outlet acted as collector electrodes. A maximum streaming potential of 2.6 V, a current of 1.3 µA, and a maximum power density of 4.3 µW cm-2 were obtained for this gold-nanoparticle-embedded PDMS channel with ultrapure water as the working fluid at an inlet pressure of 1 bar. The generated power density here was ∼256 times higher than that for the PDMS channel array without gold nanoparticles using ultrapure water as the working fluid, confirming the benefit of gold nanoparticles in the channel array, which may have potential applications in microwatt-powered lab-on-chip devices.

Gold nanoparticle-assisted enhancement in the anti-cancer properties of theaflavin against human ovarian cancer cells.

Redox-active quinones have been reported to show good potential for biological activities, while efforts are directed to explore the usefulness of these materials further in cancer management. Our previous study demonstrated that theaflavin and theaflavin-gallates (tea-extracted polyphenols) selectively induce apoptosis of tumour cells in vitro, but its concentration for showing half-maximal therapeutic response remains a matter of concern. In this report, we demonstrated that if theaflavin is conjugated with gold nanoparticles (AuNPs) to form a nanoconjugate AuNP@TfQ, its apoptotic ability increases significantly in comparison to the bare theaflavin (Tf). The nanoconjugate is prepared by following a one-step green synthesis ̶ a reaction between HAuCl4 and the aflavin at room temperature. AuNP@TfQ is characterized using particle size analysis, FESEM, UV-vis, FTIR, fluorescence, and X-ray photoelectron spectroscopytechniques. We assume that the enhanced anti-cancer effect of AuNP@TfQ appears due to the facile oxidation of the pristine theaflavin to its quinone derivative on the surface of AuNPs. The presence of quinone motif in AuNP@TfQ induces an increased level of ROS generation probably through the depolarization of mitochondria and resulted in the caspase-mediated apoptotic cell death which may hold the potential for a "magic bullet"-mediated ovarian cancer treatment.

Ethylenediamine assisted functionalization of self-organized poly (d, l-lactide-co-glycolide) patterned surface to enhance cancer cell isolation.

Protein functionalized micro-scale patterned structures are developed using a biocompatible polymer PLGA (poly (d, l-lactide-co-glycolide)) via thin film dewetting and by step-wise chemical conjugations with EDA (ethylenediamine) and anti-EpCAM (Epithelial Cell Adhesion Molecule) antibodies to target the epithelial cell adhesion molecules of cancer cells. The effectiveness of such protein functionalized patterned surface is checked through cell isolation process using blood samples spiked with different cancer cells such as MCF-7, A549, MDA-MB-231. An efficient capture yield of 92% is obtained with MCF-7 cells over a two hour incubation time. The study demonstrates the effects of cell concentration and incubation time on the binding of cancer cells to the modified patterned surfaces. For the first time, a simple and inexpensive method is reported to fabricate functionalized PLGA patterned surface for an efficient isolation of cancer cells from diluted blood samples. The method shows the potential to be used as an effective platform for the development of an improved circulating tumor cell (CTC) isolation device from the clinical blood sample.

A novel approach to fabricate dye-encapsulated polymeric micro- and nanoparticles by thin film dewetting technique.

A new method is reported for fabrication of polymeric micro- and nanoparticles from an intermediate patterned surface originated by dewetting of a polymeric thin film. Poly (d, l-lactide-co-glycolide) or PLGA, a biocompatible polymer is used to develop a thin film over a clean glass substrate which dewets spontaneously in the micro-/nano-patterned surface of size range 50nm to 3.5µm. Since another water-soluble polymer, poly vinyl alcohol (PVA) is coated on the same glass substrate before PLGA thin film formation, developed micro-/nano-patterns are easily extracted in water in the form of micro- and nanoparticle mixture of size range 50nm to 3.0µm. This simplified method is also used to effectively encapsulate a dye molecule, rhodamine B inside the PLGA micro-/nanoparticles. The developed dye-encapsulated nanoparticles, PLGA-rhodamine are separated from the mixture and tested for in-vitro delivery application of external molecules inside human lung cancer cells. For the first time, the use of thin film dewetting technique is reported as a potential route for the synthesis of polymeric micro-/nanoparticles and effective encapsulation of external species therein.

Energy generation from water flow over a reduced graphene oxide surface in a paper-pencil device.

Energy generation using liquid movement over a graphene surface generally demands a very high rate of flow (e.g.∼100 ml min(-1)). In addition, a continuous flow of liquid is unable to generate a desired voltage, as it needs modification of the substrate such as development of nanopores and criss-cross network structures. Here, we report an ultra-low-cost yet highly efficient portable device for energy conversion, by exploiting the capillary flow of an electrolyte on a filter paper matrix in which a naturally deposited gradient of reduced graphene oxide is induced through chemical synthesis. In addition, the fibres and pores present in the paper offer a criss-cross network, acting as a natural splitter of a continuous flow into tiny droplets. Our methodology thus obviates the need for any elaborate procedure for pre-generation of droplets. Further, we fabricate the necessary electrodes on filter paper by simply scribing pencil tips on the same filter paper, which facilitates the necessary electrochemical reactions. Effectively, at the anode, electrons are released, which travel through the outer circuit for cation reduction at the cathode and deliver an electrical output (voltage/current), realizing the conversion of the chemical form of energy to the electrical form in the filter paper. An absorbent pad at the channel outlet ensures continuous flow of fresh solution in the device, resulting in an inexpensive platform for power generation over a prolonged period of time. A maximum current density of 325 mA cm(-2) and a maximum power density of 53 mW cm(-2) have been observed, which significantly outweigh the capabilities of other reported devices fabricated for similar purposes.

A paper based microfluidic device for easy detection of uric acid using positively charged gold nanoparticles.

A paper based microfluidic device is fabricated that can rapidly detect very low concentrations of uric acid (UA) using 3,5,3',5'-tetramethyl benzidine (TMB), H2O2 and positively charged gold nanoparticles ((+)AuNPs). In the presence of (+)AuNPs, H2O2 reacts with TMB to produce a bluish-green colour which becomes colourless on reaction with UA. This colorimetric method can detect as low as 8.1 ppm of UA within <20 minutes on white filter paper. This technique provides an alternative way for UA detection.

Selective X-ray contrast enhancement of the spleen of living mice mediated by gold nanorods.

Gold nanomaterials (AuNPs) represent a promising new class of contrast agents for X-ray computed tomographic (CT) imaging in both research and clinical settings. These materials exhibit superior X-ray absorption properties compared with other iodinated agents, and thus require lower injection doses. Gold is nonimmunogenic and therefore contributes to safety profile in living specimens. Unfortunately, most reports on the use of AuNPs as X-ray CT enhancers only demonstrate marginal enhancement of the intended anatomical structure. In this study, we demonstrate the dramatic properties of gold nanorods (GNR) to serve as robust X-ray CT contrast-enhancing agent for selective imaging of the spleen. These organ-specific uptake properties were delineated by performing longitudinal CT imaging of living mice that were dosed with GNR at 2 day intervals. Rapid uptake in spleen was noted within 12 h of first systemic administration with a change in contrast enhancement of 90 Hounsfield units (ΔHU = 90) and with two subsequent injections a total contrast enhancement of over 200 HU was observed. The resulting images provide excellent contrast that will enable the detailed anatomical visualization and study of a range of pre-clinical models of spleen disease including infection and cancer.

Controlled splitting and focusing of a stream of nanoparticles in a converging-diverging microchannel.

We demonstrate the potential of a converging-diverging microchannel to split a stream of nanoparticles towards the interfacial region of the dispersed and the carrier phases, introduced through the middle inlet and through the remaining two inlets respectively, while maintaining a low Reynolds number limit (<10) for the flow of both phases. In addition to the splitting of passive tracer particles, such as polystyrene beads as used herein, the present setup has the potential to be utilized for a controlled reaction and thereby the separation of products towards an intended location, as observed from the experimentation with silver-nanoparticles and hydrogen-peroxide solution. Moreover, the microscale dimension of the channel allows controlled deposition of the reaction product over the bottom surface of the channel, allowing the possibility of bottom-up fabrication of microscale features. We unveil the underlying hydrodynamics that lead to such behaviours through numerical simulations, which are consistent with the experimental observations. The phenomenological features are found to be guided by the splitting of the intrinsic streamlines conforming to the flow geometry under consideration.

Bombesin peptide conjugated gold nanocages internalize via clathrin mediated endocytosis.

The nature of interaction and mechanism of internalization of receptor-avid peptide nanoparticles with cells is not yet completely understood. This article describes the cellular internalization mechanism and intracellular trafficking of peptide conjugated receptor targeted porous Gold nanocages (AuNCs) in cancer cells. We synthesized and characterized a library of AuNCs conjugated with bombesin (BBN) peptide. Evidence of selective affinity of AuNC-BBN toward gastrin releasing peptide receptors (GRPR) was obtained using radiolabeled competitive cell binding assay. Endocytic mechanism was investigated using cell inhibitor studies and monitored using optical and transmission electron microscopy (TEM). Results show AuNC-BBN uptake in PC3 cells is mediated by clathrin mediated endocytosis (CME). Indeed, in the presence of CME inhibitors, AuNC-BBN uptake in cells is reduced up to 84%. TEM images further confirm CME characteristic clathrin coated pits and lysosomal release of AuNCs. These results demonstrate that peptide ligands conjugated to the surface of nanoparticles maintain their target specificity. This bolsters the case for peptide robustness and its persisting functionality in intracellular vehicular delivery systems.

Gold nanoparticle based X-ray contrast agent for tumor imaging in mice and dog: a potential nano-platform for computer tomography theranostics.

The goal of our study was to demonstrate the utility of nanocrystalline gold as an X-ray contrast agent for imaging tumor in living subjects. Even though significant progress has been achieved in this area by researchers, clinical translation remains challenging. Here, we investigated biocompatible gum Arabic stabilized gold nanocrystals (GA-AuNPs) as X-ray contrast agent in tumor bearing mice and dog. Single intratumoral injections of GA-AuNP resulted in X-ray contrast change of -26 HU in the tumor region after 1 hour post-injection period. Subsequently, five intratumoral injections were performed in the mice. The change in CT number in tumor region is not progressive; rather it reaches a saturation point after fourth injection. These data suggested that accumulation of GA-AuNP reaches a threshold limit within a short time period (5 h), and is retained in the tumor tissue for the rest of the period of investigation. A pilot study was conducted in a client-owned dog presented with collision tumor of thyroid carcinoma and osteosarcoma. In this study, GA-AuNP was injected intratumorally in dog and a contrast enhancement of 12 deltaHU was observed. The CT images of both mice and dog clearly demonstrated that GA-AuNP was effectively distributed and retained throughout the tumor site. The CT data obtained by the present study would provide the crucial dosimetry information for strategic therapy planning using this construct. Both mice and dog did not show any clinical changes, thereby confirming that GA-AuNP did not induce toxicity and can be explored for future clinical applications.

A paper based self-pumping and self-breathing fuel cell using pencil stroked graphite electrodes.

We develop a paper based fuel cell in which fluids flow through a capillary transport mechanism. The pencil stroked graphite electrodes take oxygen from quiescent air. This simple and efficient paper fuel cell can generate energy to the tune of 32 mW cm(-2) over a prolonged duration of around 1000 minutes, and with the consumption of a very low volume of formic acid as fuel (~1 mL).

Nanomaterial processing using self-assembly-bottom-up chemical and biological approaches.

Nanotechnology is touted as the next logical sequence in technological evolution. This has led to a substantial surge in research activities pertaining to the development and fundamental understanding of processes and assembly at the nanoscale. Both top-down and bottom-up fabrication approaches may be used to realize a range of well-defined nanostructured materials with desirable physical and chemical attributes. Among these, the bottom-up self-assembly process offers the most realistic solution toward the fabrication of next-generation functional materials and devices. Here, we present a comprehensive review on the physical basis behind self-assembly and the processes reported in recent years to direct the assembly of nanoscale functional blocks into hierarchically ordered structures. This paper emphasizes assembly in the synthetic domain as well in the biological domain, underscoring the importance of biomimetic approaches toward novel materials. In particular, two important classes of directed self-assembly, namely, (i) self-assembly among nanoparticle-polymer systems and (ii) external field-guided assembly are highlighted. The spontaneous self-assembling behavior observed in nature that leads to complex, multifunctional, hierarchical structures within biological systems is also discussed in this review. Recent research undertaken to synthesize hierarchically assembled functional materials have underscored the need as well as the benefits harvested in synergistically combining top-down fabrication methods with bottom-up self-assembly.