Microneedle-based nanoporous gold electrochemical sensor for real-time catecholamine detection.

Dopamine (DA), epinephrine (EP), and norepinephrine (NEP) are the main catecholamine of clinical interest, as they play crucial roles in the regulation of nervous and cardiovascular systems and are involved in some brain behaviors, such as stress, panic, anxiety, and depression. Therefore, there is an urgent need for a reliable sensing device able to provide their continuous monitoring in a minimally invasive manner. In this work, the first highly nanoporous gold (h-nPG) microneedle-based sensor is presented for continuous monitoring of catecholamine in interstitial fluid (ISF). The h-nPG microneedle-based gold electrode was prepared by a simple electrochemical self-templating method that involves two steps, gold electrodeposition and hydrogen bubbling at the electrode surface, realized by sweeping the potential between + 0.8 V and 0 V vs Ag/AgCl for 25 scans in a 10 mM HAuCl4 solution containing 2.5 M NH4Cl, and successively applying a fixed potential of - 2 V vs Ag/AgCl for 60 s. The resulting microneedle-based h-nPG sensor displays an interference-free total catecholamine detection expressed as NEP concentration, with a very low LOD of 100 nM, excellent sensitivity and stability, and fast response time (< 4 s). The performance of the h-nPG microneedle array sensor was successively assessed in artificial ISF and in a hydrogel skin model at typical physiological concentrations.

Autocatalytic Metallization of Fabrics Using Si Ink, for Biosensors, Batteries and Energy Harvesting.

Commercially available metal inks are mainly designed for planar substrates (for example, polyethylene terephthalate foils or ceramics), and they contain hydrophobic polymer binders that fill the pores in fabrics when printed, thus resulting in hydrophobic electrodes. Here, a low-cost binder-free method for the metallization of woven and nonwoven fabrics is presented that preserves the 3D structure and hydrophilicity of the substrate. Metals such as Au, Ag, and Pt are grown autocatalytically, using metal salts, inside the fibrous network of fabrics at room temperature in a two-step process, with a water-based silicon particle ink acting as precursor. Using this method, (patterned) metallized fabrics are being enabled to be produced with low electrical resistance (less than 3.5 Ω sq-1). In addition to fabrics, the method is also compatible with other 3D hydrophilic substrates such as nitrocellulose membranes. The versatility of this method is demonstrated by producing coil antennas for wireless energy harvesting, Ag-Zn batteries for energy storage, electrochemical biosensors for the detection of DNA/proteins, and as a substrate for optical sensing by surface enhanced Raman spectroscopy. In the future, this method of metallization may pave the way for new classes of high-performance devices using low-cost fabrics.

Microneedle Array-Based Platforms for Future Theranostic Applications.

Theranostics involves finding the biomarkers of a disease, fighting them through site specific drug delivery and following them for prognosis of the disease. Microneedle array technology has been used for drug delivery and extended for continuous monitoring of analytes present in the skin compartment. We envisage the use of microneedle arrays for future theranostic applications. The potential of combining microneedle array-based drug delivery and diagnostics as part of closed-loop control system for the management of diseases and delivery of precision drugs in individual patients is reported in this paper.

Polyfunctionalised Nanoparticles Bearing Robust Gadolinium Surface Units for High Relaxivity Performance in MRI.

The first example of an octadentate gadolinium unit based on DO3A (hydration number q=1) with a dithiocarbamate tether has been designed and attached to the surface of gold nanoparticles (around 4.4 nm in diameter). In addition to the superior robustness of this attachment, the restricted rotation of the Gd complex on the nanoparticle surface leads to a dramatic increase in relaxivity (r1 ) from 4.0 mm-1 s-1 in unbound form to 34.3 mm-1 s-1 (at 10 MHz, 37 °C) and 22±2 mm-1 s-1 (at 63.87 MHz, 25 °C) when immobilised on the surface. The one-pot synthetic route provides a straightforward and versatile way of preparing a range of multifunctional gold nanoparticles. The incorporation of additional surface units for biocompatibility (PEG and thioglucose units) and targeting (folic acid) leads to little detrimental effect on the high relaxivity observed for these non-toxic multifunctional materials. In addition to the passive targeting attributed to gold nanoparticles, the inclusion of a unit capable of targeting the folate receptors overexpressed by cancer cells, such as HeLa cells, illustrates the potential of these assemblies.

Development of drug-loaded protein nanoparticles displaying enzymatically-conjugated DNA aptamers for cancer cell targeting.

Modification of protein-based drug carriers with tumor-targeting properties is an important area of research in the field of anticancer drug delivery. To this end, we developed nanoparticles comprised of elastin-like polypeptides (ELPs) with fused poly-aspartic acid chains (ELP-D) displaying DNA aptamers. DNA aptamers were enzymatically conjugated to the surface of the nanoparticles via genetic incorporation of Gene A* protein into the sequence of the ELP-D fusion protein. Gene A* protein, derived from bacteriophage ϕX174, can form covalent complexes with single-stranded DNA via the latter's recognition sequence. Gene A* protein-displaying nanoparticles exhibited the ability to deliver the anticancer drug paclitaxel (PTX), whilst retaining activity of the conjugated Gene A* protein. PTX-loaded protein nanoparticles displaying DNA aptamers known to bind to the MUC1 tumor marker resulted in increased cytotoxicity with MCF-7 breast cancer cells compared to PTX-loaded protein nanoparticles without the DNA aptamer modification.

Dual Recognition Element Lateral Flow Assay Toward Multiplex Strain Specific Influenza Virus Detection.

Different influenza virus strains have caused a number of recent outbreaks killing scores of people and causing significant losses in animal farming. Simple, rapid, sensitive, and specific detection of particular strains, such as a pandemic strain versus a previous seasonal influenza, plays a crucial role in the monitoring, controlling, and management of outbreaks. In this paper we describe a dual recognition element lateral flow assay (DRELFA) which pairs a nucleic acid aptamer with an antibody for use as a point-of-care platform which can detect particular strains of interest. The combination is used to overcome the individual limitations of antibodies' cross-reactivity and aptamers' slow binding kinetics. In the detection of influenza viruses, we show that DRELFA can discriminate a particular virus strain against others of the same subtype or common respiratory diseases while still exhibiting fast binding kinetic of the antibody-based lateral flow assay (LFA). The improvement in specificity that DRELFA exhibits is an advantage over the currently available antibody-based LFA systems for influenza viruses, which offer discrimination between influenza virus types and subtypes. Using quantitative real-time PCR (qRT-PCR), it showed that the DRELFA is very effective in localizing the analyte to the test line (consistently over 90%) and this is crucial for the sensitivity of the device. In addition, color intensities of the test lines showed a good correlation between the DRELFA and the qRT-PCR over a 50-fold concentration range. Finally, lateral flow strips with a streptavidin capture test line and an anti-antibody control line are universally applicable to specific detection of a wide range of different analytes.

Evaluation of a minimally invasive glucose biosensor for continuous tissue monitoring.

We describe here a minimally invasive glucose biosensor based on a microneedle array electrode fabricated from an epoxy-based negative photoresist (SU8 50) and designed for continuous measurement in the dermal compartment with minimal pain. These minimally invasive, continuous monitoring sensor devices (MICoMS) were produced by casting the structures in SU8 50, crosslinking and then metallising them with platinum or silver to obtain the working and reference electrodes, respectively. The metallised microneedle array electrodes were subsequently functionalised by entrapping glucose oxidase in electropolymerised polyphenol (PP) film. Sensor performance in vitro showed that glucose concentrations down to 0.5 mM could be measured with a response times (T90) of 15 s. The effect of sterilisation by Co60 irradiation was evaluated. In preparation for further clinical studies, these sensors were tested in vivo in a healthy volunteer for a period of 3-6 h. The sensor currents were compared against point measurements obtained with a commercial capillary blood glucometer. The epoxy MICoMS devices showed currents values that could be correlated with these. Graphical Abstract Microneedle arrays for continuous glucose monitoring in dermal interstitial fluid.

Enzymatic cyanide degradation by cell-free extract of Rhodococcus UKMP-5M.

The cell-free extract of locally isolated Rhodococcus UKMP-5M strain was used as an alternative to develop greener and cost effective cyanide removal technology. The present study aims to assess the viability of the cell-free extract to detoxify high concentrations of cyanide which is measured through the monitoring of protein concentration and specific cyanide-degrading activity. When cyanide-grown cells were subjected to grinding in liquid nitrogen which is relatively an inexpressive and fast cell disruption method, highest cyanide-degrading activity of 0.63 mM min(-1) mg(-1) protein was obtained in comparison to enzymatic lysis and agitation with fine glass beads. The cell-free extracts managed to degrade 80% of 20 mM KCN within 80 min and the rate of cyanide consumption increased linearly as the concentration of protein was raised. In both cases, the addition of co-factor was not required which proved to be advantageous economically. The successful formation of ammonia and formate as endproducts indicated that the degradation of cyanide by Rhodococcus UKMP-5M proceeded via the activity of cyanidase and the resulting non-toxic products are safe for disposal into the environment. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Thus, the utilization of cell-free extracts as an alternative to live microbial in cyanide degradation offers numerous advantageous such as the potential to tolerate and degrade higher concentration of cyanide and total reduction in the overall cost of operation since the requirement for nutrient support is irrelevant.

High efficiency acetylcholinesterase immobilization on DNA aptamer modified surfaces.

We report here the in vitro selection of DNA aptamers for electric eel acetylcholinesterase (AChE). One selected aptamer sequence (R15/19) has a high affinity towards the enzyme (Kd=157±42 pM). Characterization of the aptamer showed its binding is not affected by low ionic strength (~20 mM), however significant reduction in affinity occurred at high ionic strength (~1.2 M). In addition, this aptamer does not inhibit the catalytic activity of AChE that we exploit through immobilization of the DNA on a streptavidin-coated surface. Subsequent immobilization of AChE by the aptamer results in a 4-fold higher catalytic activity when compared to adsorption directly on to plastic.

A low-volume LC/MS method for highly sensitive monitoring of phenoxymethylpenicillin, benzylpenicillin, and probenecid in human serum.

Background: The optimization of antimicrobial dosing plays a crucial role in improving the likelihood of achieving therapeutic success while reducing the risks associated with toxicity and antimicrobial resistance. Probenecid has shown significant potential in enhancing the serum exposure of phenoxymethylpenicillin, thereby allowing for lower doses of phenoxymethylpenicillin to achieve similar pharmacokinetic/pharmacodynamic (PK/PD) targets. We developed a triple quadrupole liquid chromatography mass spectrometry (TQ LC/MS) analysis of, phenoxymethylpenicillin, benzylpenicillin and probenecid using benzylpenicillin-d7 and probenecid-d14 as IS in single low-volumes of human serum, with improved limit of quantification to support therapeutic drug monitoring. Methods: Sample clean-up was performed by protein precipitation using acetonitrile. Reverse phase chromatography was performed using TQ LC/MS. The mobile phase consisted of 55% methanol in water + 0.1% formic acid, with a flow rate of 0.4 mL min-1. Antibiotic stability was assessed at different temperatures. Results: Chromatographic separation was achieved within 2 minutes, allowing simultaneous measurement of phenoxymethylpenicillin, benzylpenicillin and probenecid in a single 15 µL blood sample. Validation indicated linearity over the range 0.0015-10 mg L-1, with accuracy of 96-102% and a LLOQ of 0.01 mg L-1. All drugs demonstrated good stability under different storage conditions. Conclusion: The developed method is simple, rapid, accurate and clinically applicable for the quantification of phenoxymethylpenicillin, benzylpenicillin and probenecid in tandem.

Robotic APTamer-Enabled Electrochemical Reader (RAPTER) System for Automated Aptamer-Mediated Electrochemical Analysis.

Electrochemical aptamer-based (E-AB) sensors using conformational change-induced electron transfer kinetics are sensitive, reagent-less, and cost-effective tools for molecular sensing. Current advances in this technology can allow continuous drug pharmacokinetic monitoring in living animals (Dauphin-Ducharme et al., ACS Sens 4(10):2832-2837, 2019; Idili et al., Chem Sci 10(35):8164-8170, 2019), as well as automated analysis of hormone pulsatility (Liang et al., Nat Commun 10(1):852, 2019). In this chapter, we provide the methodology for an automated E-AB conformational change-based robotic sensing platform. By using an open-source programmable robotic system, this method can be adapted to a wide range of experimental scenarios.

A sprayed graphene transistor platform for rapid and low-cost chemical sensing.

We demonstrate a novel and versatile sensing platform, based on electrolyte-gated graphene field-effect transistors, for easy, low-cost and scalable production of chemical sensor test strips. The Lab-on-PCB platform is enabled by low-boiling, low-surface-tension sprayable graphene ink deposited on a substrate manufactured using a commercial printed circuit board process. We demonstrate the versatility of the platform by sensing pH and Na+ concentrations in an aqueous solution, achieving a sensitivity of 143 ± 4 µA per pH and 131 ± 5 µA per log10Na+, respectively, in line with state-of-the-art graphene chemical sensing performance.

Triple quadrupole LC/MS method for the simultaneous quantitative measurement of cefiderocol and meropenem in serum.

Background: therapeutic drug monitoring is a crucial aspect of the management of hospitalized patients. The correct dosage of antibiotics is imperative to ensure their adequate exposure specially in critically ill patients. The aim of this study is to establish and validate a robust and fast liquid chromatography-tandem mass spectrometry (LC/MS) method for the simultaneous quantification of two important antibiotics in critically ill patients, cefiderocol and meropenem in human plasma. Methods: sample clean-up was performed by protein precipitation using acetonitrile. Reverse phase chromatography was performed using triple quadrupole LC/MS. The mobile phase was consisted of 55% methanol in water +0.1% formic acid, with flow rate of 0.4 ml min-1. Antibiotics stability was assessed at different temperatures. Serum protein binding was assessed using ultrafiltration devices. Results: chromatographic separation was achieved within 1.5 minutes for all analytes. Validation has demonstrated the method to be linear over the range 0.0025-50 mg L-1 for cefiderocol and 0.00028-50 mg L-1 for meropenem, with accuracy of 94-101% and highly sensitive, with LLOQ ≈ 0.02 mg L-1 and 0.003 mg L-1 for cefiderocol and meropenem, respectively. Both cefiderocol and meropenem showed a good stability at room temperature over 6 h, and at (4 °C) over 24 h. Cefiderocol and meropenem demonstrated a protein binding of 49-60% and 98%, respectively in human plasma. Conclusion: the developed method is simple, rapid, accurate and clinically applicable for the quantification of cefiderocol and meropenem.

A rapid, simple, high-performance liquid chromatography method for the clinical measurement of beta-lactam antibiotics in serum and interstitial fluid.

Background: enhanced methods of therapeutic drug monitoring are required to support the individualisation of antibiotic dosing based on pharmacokinetics (PK) parameters. PK studies can be hampered by limited total serum volume, especially in neonates, or by sensitivity in the case of critically ill patients. We aimed to develop a liquid chromatography-mass spectrometry (LC/MS) analysis of benzylpenicillin, phenoxymethylpenicillin and amoxicillin in single low volumes of human serum and interstitial fluid (ISF) samples, with an improved limit of detection (LOD) and limit of quantification (LOQ), compared with previously published assays. Methods: sample clean-up was performed by protein precipitation using acetonitrile. Reverse phase chromatography was performed using triple quadrupole LC/MS. The mobile phase consisted of 55% methanol in water + 0.1% formic acid, with a flow rate of 0.4 mL min-1. Antibiotics stability was assessed at different temperatures. Results: chromatographic separation was achieved within 3 minutes for all analytes. Three common penicillins can now be measured in a single low-volume blood and ISF sample (15 µL) for the first time. Validation has demonstrated the method to be linear over the range 0.0015-10 mg L-1, with an accuracy of 93-104% and high sensitivity, with LOD ≈ 0.003 mg L-1 and LOQ ≈ 0.01 mg L-1 for all three analytes, which is critical for use in dose optimisation/individualisation. All evaluated penicillins indicated good stability at room temperature over 4 h, at (4 °C) over 24 h and at -80 °C for 6 months. Conclusion: the developed method is simple, rapid, accurate and clinically applicable for the quantification of three penicillin classes.

Continuous Measurement of Lactate Concentration in Human Subjects through Direct Electron Transfer from Enzymes to Microneedle Electrodes.

Microneedle lactate sensors may be used to continuously measure lactate concentration in the interstitial fluid in a minimally invasive and pain-free manner. First- and second-generation enzymatic sensors produce a redox-active product that is electrochemically sensed at the electrode surface. Direct electron transfer enzymes produce electrons directly as the product of enzymatic action; in this study, a direct electron transfer enzyme specific to lactate has been immobilized onto a microneedle surface to create lactate-sensing devices that function at low applied voltages (0.2 V). These devices have been validated in a small study of human volunteers; lactate concentrations were raised and lowered through physical exercise and subsequent rest. Lactazyme microneedle devices show good agreement with concurrently obtained and analyzed serum lactate levels.

Biosensors Based on Acetylcholinesterase Immobilized on Clay-Gold Nanocomposites for the Discrimination of Chlorpyrifos and Carbaryl.

This work aims at evaluating a utilization of diverse clay mineral/gold nanoparticles/acetylcholinesterase (clay/AuNPs/AChE) biosensors by using principal component analysis (PCA) for the discrimination of pesticide types and their concentration levels both in the synthetic and real samples. Applications of simple and low-cost clay/AuNP composites of different characteristics as modified-electrode materials are highlighted. Four types of clay minerals, namely, platelike kaolinite (Kaol: 1:1 aluminum phyllosilicate), globular montmorillonite (Mt: 2:1 aluminum phyllosilicate), globular bentonite (Bent: 2:1 aluminum phyllosilicate), and fibrous sepiolite (Sep: 2:1 inverted ribbons of magnesium phyllosilicate), were selected as the base materials. Due to the distinct characteristics of the selected clay, the derived clay/AuNP composites resulted in different physical morphologies, AuNP sizes and loadings, matrix hydrophobicity, and active AChE loading per electrode. These, in turn, caused divergent electrochemical responses for the pesticide determination; hence, no other enzymes apart from AChE were necessary for the fabrication of distinct biosensors. Physical and chemical characterizations of clay/AuNPs were conducted using scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy techniques. The electrochemical information was recorded by cyclic voltammetry and amperometry techniques. The enzyme inhibition results obtained from the pesticides were treated and used as input data to obtain PCA results. The four fabricated clay/AuNPs/AChE biosensors were able to discriminate chlorpyrifos and carbaryl and their concentration levels for synthetic pesticides and real samples. It was disclosed that a high enzyme inhibition and a high hydrophobic modified-electrode material affect a highly sensitive pesticide biosensor. The hydrophobic/hydrophilic character of the modified-electrode material plays a major role in discriminating the pesticide types and their concentration levels by the proposed single-enzyme sensor system. The PCA results illustrated that PC2 described the different types of pesticides, and PC1 showed the level of pesticide concentration with high first two principal components. The mixed pesticides could be identified at an especially low total concentration of 0.5 ng/mL in real samples.

A simple method for controlled immobilization of proteins on modified SAMs.

This paper describes a method for modifying self-assembled monolayers (SAMs) with the nitrilotriacetic acid (NTA) group for subsequent immobilization of hexahistidine tagged proteins. The method has two important improvements over previous ones; firstly it avoids the need to carry out a complex synthesis of the chelator alkanethiols prior to deposition because the reactions are performed in situ on a preassembled SAM. This in situ approach also avoids phase segregation of alkanethiols with different functional groups, especially bulky ones such as NTA and tri(ethylene glycol), since a simple SAM is employed as the starting material. The approach reported here uses mercaptohexadecanoic acid to form a well-ordered homogeneous carboxyl-terminated SAM on a gold surface. The carboxyl group was then condensed with an NTA derivative containing an amino group to form a peptide bond. The product is a surface that, after chelating Ni(2+) ions, binds histidine tagged proteins. The loading of NTA groups can be controlled by choice of reaction conditions thereby removing the need for a second alkanethiol to dilute the surface density of chelator groups and prevent molecular crowding. Both factors allow rapid attainment of optimal protein loading. Fluorescence imaging demonstrated that (His)(6) enhanced green fluorescent protein was reversibly immobilized and importantly, was functional on the surface. Furthermore, data from surface plasmon resonance, cyclic voltammetry and fluorescence spectrometry provided additional information on the specific and reversible immobilization of (His)(6) proteins on the NTA-modified SAM surface.

CE-based sample quality assessment prior to 2-D gel electrophoresis: towards the standardization of gel-based proteomics.

2-DE remains one of the most commonly used separation techniques for complex protein mixtures. This article describes a new approach to 2-DE sample assessment using SDS capillary gel electrophoresis (in Beckman Coulter sieving medium) combined with multi-pixel detection. The performance of this platform was investigated using protein samples prepared for 2-DE. The capability to characterize 2-DE sample was tested and the results show that the repeatability of peak migration time and intensity are better than 2% RSD. The system gives good resolution, accurate molecular mass assignment, as well as absolute and relative quantification of proteins. Notably, this study also demonstrates the use of this platform to screen the quality of simple and complex 2-DE samples. Implementation of this technique in the proteomics workflow will not only improve the success rate of 2-DE, but will also enable sample verification before 2-DE and allow the relative quantification of proteins. The validation of differential protein expression is also demonstrated using the combined information of relative molecular mass and relative quantification. It is the first time that a rapid and visual evaluation method is reported for the quality assessment of 2-DE samples.

Decolourization and biodegradation of azo dye methyl red by Rhodococcus strain UCC 0016.

In the present study, locally isolated Rhodococcus strains were attempted as biological tools for methyl red removal, a mutagenic azo dye posing threat to the environment if left untreated. Rhodococcus strain UCC 0016 demonstrated superior methyl red-decolourizing activity of 100% after 24 h at static condition in comparison to Rhodococcus strain UCC 0008 which recorded 65% decolourization after 72 h. Optimization of physicochemical parameters at 30°C, pH 7 and supplementing glucose as the carbon source resulted in improved methyl red-decolourizing activity at static condition and reduced the time taken to achieve complete decolourization by 80%. Higher concentration of methyl red (5 g/L) was able to be decolourized completely within 10 h by adopting the technology of immobilization. The encapsulated cells of Rhodococcus strain UCC 0016 demonstrated higher substrate affinity (Km = 0.6995 g/L) and an accelerated rate of disappearance of methyl red (Vmax = 0.3203 g/L/h) compared to the free cells. Furthermore, the gellan gum beads could be reused up to nine batches without substantial loss in the catalytic activity indicating the economic importance of this protocol. Analysis of methyl red degradation products revealed no germination inhibition on Triticum aestivum and Vigna radiata demonstrating complete toxicity removal of the parent dye after biological treatment. The occurrence of new and altered peaks (UV-Vis and FTIR) further supported the notion that the removal of methyl red by Rhodococcus strain UCC 0016 was indeed through biodegradation. Therefore, this strain has a huge potential as a candidate for efficient bioremediation of wastewater containing methyl red.

Antioxidant lipoic acid ligand-shell gold nanoconjugates against oxidative stress caused by α-synuclein aggregates.

Gold nanoparticles are becoming a promising platform for the delivery of drugs to treat neurodegenerative diseases. Parkinson's disease, associated with the aggregation of α-synuclein, is a condition that results in dysfunctional neuronal cells leading to their degeneration and death. Oxidative stress has been strongly implicated as a common feature in this process. The limited efficacy of the traditional therapies and the development of associated severe side effects present an unmet need for preventive and adjuvant therapies. The organosulfur compound lipoic acid, naturally located in the mitochondria, plays a powerful antioxidative role against oxidative stress. However, the efficacy is limited by its low physiological concentration, and the administration is affected by its short half-life and bioavailability due to hepatic degradation. Here we exploited the drug delivery potential of gold nanoparticles to assemble lipoic acid, and administered the system into SH-SY5Y cells, a cellular model commonly used to study Parkinson's disease. We tested the nanoconjugates of GNPs-LA, under an oxidative environment induced by gold nanoparticle/α-synuclein conjugates (GNPs-α-Syn). GNPs-LA were found to be biocompatible and capable of restoring the cell damage caused by high-level reactive oxygen species generated by excessive oxidative stress in the cellular environment. We conclude that GNPs-LA may serve as promising drug delivery vehicles conveying antioxidant molecules for the treatment of Parkinson's disease.