Correction: Musa et al. An Electrochemical Screen-Printed Sensor Based on Gold-Nanoparticle-Decorated Reduced Graphene Oxide-Carbon Nanotubes Composites for the Determination of 17-ß Estradiol. Biosensors 2023, 13, 491.

In the original publication [...].

A non-faradaic impedimetric biosensor for monitoring of caspase 9 in mammalian cell culture.

Lower yields and poorer quality of biopharmaceutical products result from cell death in bioreactors. Such cell death is commonly associated with programmed cell death or apoptosis. During apoptosis, caspases are activated and cause a cascade of events that eventually lead to cell destruction. We report on an impedance spectroscopy measurement technique for the detection of total caspase-9 in buffer and complex fluids, such as cell culture media. Enhanced sensitivity is achieved by leveraging the physiochemical properties of zinc oxide and copper oxide at the electrode-solution interface. Characterisation of the biosensor surface was performed using scanning electron microscopy and indirectly using an enzyme-linked immunosorbent assay. The characteristic biomolecular interactions between the target analyte and specific capture probe of the biosensor are quantified using non-faradaic electrical impedance spectroscopy (nfEIS). The proof-of-concept biosensor demonstrated a detection limit of 0.07 U/mL (0.032 µM) in buffer. The sensor requires a low sample volume of 50 µL without the need for sample dilution facilitating rapid analysis. Using a luminescence-based assay, the presence of active caspase-9 was detected in the culture media following exposure to a pro-apoptotic agent. We envision that the caspase-9 biosensor will be useful as a cell stress screening device for apoptosis monitoring.

An Electrochemical Screen-Printed Sensor Based on Gold-Nanoparticle-Decorated Reduced Graphene Oxide-Carbon Nanotubes Composites for the Determination of 17-ß Estradiol.

In this study, a screen-printed electrode (SPE) modified with gold-nanoparticle-decorated reduced graphene oxide-carbon nanotubes (rGO-AuNPs/CNT/SPE) was used for the determination of estradiol (E2). The AuNPs were produced through an eco-friendly method utilising plant extract, eliminating the need for severe chemicals, and remove the requirements of sophisticated fabrication methods and tedious procedures. In addition, rGO-AuNP serves as a dispersant for the CNT to improve the dispersion stability of CNTs. The composite material, rGO-AuNPs/CNT, underwent characterisation through scanning electron microscopy (SEM), ultraviolet-visible absorption spectroscopy (UV-vis), Fourier-transform infrared (FTIR) spectroscopy, and atomic force microscopy (AFM). The electrochemical performance of the modified SPE for estradiol oxidation was characterised using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques. The rGO-AuNPs/CNT/SPE exhibited a notable improvement compared to bare/SPE and GO-CNT/SPE, as evidenced by the relative peak currents. Additionally, we employed a baseline correction algorithm to accurately adjust the sensor response while eliminating extraneous background components that are typically present in voltammetric experiments. The optimised estradiol sensor offers linear sensitivity from 0.05-1.00 µM, with a detection limit of 3 nM based on three times the standard deviation (3δ). Notably, this sensing approach yields stable, repeatable, and reproducible outcomes. Assessment of drinking water samples indicated an average recovery rate of 97.5% for samples enriched with E2 at concentrations as low as 0.5 µM%, accompanied by only a modest coefficient of variation (%CV) value of 2.7%.

Modelling a dynamic magneto-agglutination bioassay.

The process of developing an end-to-end model of a magneto-immunoassay is described, simulating the agglutination effect due to the specific binding of bacteria to paramagnetic particles. After establishing the properties of the dose-specific agglutination through direct imaging, a microfluidic assay was used to demonstrate changes in the magnetophoretic transport dynamics of agglutinated clusters via transient inductive magentometer measurements. End-to-end mathematical modelling is used to establish the physical processes underlying the assay. First, a modified form of Becker-Döring nucleation kinetic equations is used to establish a relationship between analyte dose and average cluster size. Next, Stokes flow equations are used to establish a relationship between cluster size and speed of motion within the fluid chamber. This predicts a cluster-size dynamic profile of concentration of PMPs versus time when the magnetic field is switched between the two actuated magnets. Finally, inductive modelling is carried out to predict the response of the magnetometer circuit in response to the dynamics of magnetic clusters. The predictions of this model are shown to agree well with the results of experiments, and to predict the shape of the dose-response curve.

Modeling Peptide Nucleic Acid Binding Enthalpies Using MM-GBSA.

The binding enthalpies of peptide nucleic acid (PNA) homoduplexes were predicted using a molecular mechanics generalized Born surface area approach. Using the nucleic acid nearest-neighbor model, these were decomposed into sequence parameters which could replicate the enthalpies from thermal melting experiments with a mean error of 8.7%. These results present the first systematic computational investigation into the relationship between sequence and binding energy for PNA homoduplexes and identified a stabilizing helix initiation enthalpy not observed for nucleic acids with phosphoribose backbones.

Propofol detection for monitoring of intravenous anaesthesia: a review.

This paper presents a review of established and emerging methods for detecting and quantifying the intravenous anaesthetic propofol in solution. There is growing evidence of numerous advantages of total intravenous anaesthesia using propofol compared to conventional volatile-based anaesthesia, both in terms of patient outcomes and environmental impact. However, volatile-based anaesthesia still accounts for the vast majority of administered general anaesthetics, largely due to a lack of techniques for real-time monitoring of patient blood propofol concentration. Herein, propofol detection techniques that have been developed to date are reviewed alongside a discussion of remaining challenges.

Classification of the mechanism of toxicity as applied to human cell line ECV304.

The objective of this study was to identify the pattern of cytotoxicity testing of the human cell line ECV304 using three techniques of an ensemble learning algorithm (bagging, boosting and stacking). The study of cell morphology of ECV304 cell line was conducted using impedimetric measurement. Three types of toxins were applied to the ECV304 cell line namely 1 mM hydrogen peroxide (H2O2), 5% dimethyl sulfoxide and 10 µg Saponin. The measurement was conducted using electrodes and lock-in amplifier to detect impedance changes during cytotoxicity testing within a frequency range 200 and 830 kHz. The results were analysed, processed and extracted using detrended fluctuation analysis to obtain characteristics and features of the cells when exposed to the each of the toxins. Three ensemble algorithms applied showed slightly different results on the performance for classifying the data set from the feature extraction that was performed. However, the results show that the cell reaction to the toxins could be classified.

Nanoparticle-based 3D membrane for impedimetric biosensor applications.

This paper reports on a comparison between nano-ZnO/CuO and nano-ZnO nitrocellulose membrane biosensors, both of which were fabricated using a simple and inexpensive sonication technique. To produce the nano-ZnO/CuO membranes, the technique involved sonication of 1% (w/v) ZnO and 1% (w/v) CuO nano-crystal colloidal suspensions, with a volume ratio of 1:2. The membranes were analysed by scanning electron microscopy and energy-dispersive spectroscopy, which showed the gradated distribution of nanoparticles in the membrane. Impedance spectroscopy demonstrated that the sonication resulted in a greater than two-fold enhancement of the output signal. Changes in impedance phase values, at a frequency of 100 Hz, were used to establish dose dependent responses for C-reactive protein (CRP). Limits of detection of 27 pg/mL for the 1% (w/v) nano-ZnO and 16 pg/mL for the 1% (w/v) nano-ZnO/CuO nitrocellulose membrane biosensors were demonstrated.

A non-contact impedimetric biosensing system for classification of toxins associated with cytotoxicity testing.

We report on a novel impedance spectroscopy measurement and data analysis technique for cytotoxicity testing. The technique combines non-contact measurement with real-time impedance data analysis based on the toxin dose dependency of the outputs, making it suitable for high throughput screening. A multi-electrode array was designed and fabricated such that a standard well plate could be positioned above the electrodes, negating the requirement for bespoke culture wells with integrated electrodes. For cytotoxicity testing, endothelial cells, type ECV304, within the wells were exposed to various concentrations of 3 toxins, dimethyl sulphoxide, cadmium chloride and saponin, which exhibit different modes of action on cells. Impedance spectra were recorded every 30 min over a 24 h period. From the spectra 'toxin maps' were produced which presented the correlation between impedance output and dose of toxin versus frequency and time. The results demonstrated characteristic toxin maps for each toxin and significantly differences between the three toxins studied. Using complementary measurement methods, we showed that these differences in toxin maps related to morphological and physiological changes in the cells due to the differing mode of action of each toxin.

A Copper Oxide/Zinc Oxide Composite Nano-Surface for Use in a Biosensor.

In this study, biosensors based on zinc oxide⁻copper oxide composite nano-surfaces were prepared using a simple and inexpensive distributed colloidal technique. Combinations of mixed dispersions with volume ratios of 1:1, 1:2 and 2:1 ZnO:CuO were compared. The uniform nano-crystalline sensor surfaces on polyethylene terephthalate (PET) were analysed using scanning electron microscopy (SEM), Atomic Force Microscopy (AFM) and Raman Spectroscopy. The ZnO⁻CuO composite biosensor nano-surfaces showed a significantly increased impedimetric signal compared with pure ZnO nanocrystals, and the maximum output was achieved with a volume ratio of 1:2 ZnO/CuO. The antibody capture of C-reactive protein (CRP) on the nano-surfaces was used to demonstrate the enhanced signal generated with increasing amounts of CuO in the nano-surface.

Facile and inexpensive fabrication of zinc oxide based bio-surfaces for C-reactive protein detection.

The paper reports a biosensor formed from antibody coated ZnO nano-crystals which has been prepared using a rapid and inexpensive fabrication method which utilises colloidal dispersion enhanced using sonication. This technique was used to prepare highly ordered and uniform nano-crystalline sensor surfaces on polyethylene terephthalate (PET) using 0.5%, 1% and 5% concentrations of zinc oxide nano-crystal suspensions. Impedance spectroscopy was employed to interrogate the sensor surfaces and confirmed high reproducibility of the fabrication process. Changes in impedance values, at a frequency of 138 Hz, were used to establish dose dependent responses for C-reactive protein (CRP) antigen. A limit of detection of less than 1 ng/ml was demonstrated for nano-surfaces fabricated from concentrations of 1% ZnO.

An inexpensive, fast and sensitive quantitative lateral flow magneto-immunoassay for total prostate specific antigen.

We describe the detection characteristics of a device the Resonant Coil Magnetometer (RCM) to quantify paramagnetic particles (PMPs) in immunochromatographic (lateral flow) assays. Lateral flow assays were developed using PMPs for the measurement of total prostate specific antigen (PSA) in serum samples. A detection limit of 0.8 ng/mL was achieved for total PSA using the RCM and is at clinically significant concentrations. Comparison of data obtained in a pilot study from the analysis of serum samples with commercially available immunoassays shows good agreement. The development of a quantitative magneto-immunoassay in lateral flow format for total PSA suggests the potential of the RCM to operate with many immunoassay formats. The RCM has the potential to be modified to quantify multiple analytes in this format. This research shows promise for the development of an inexpensive device capable of quantifying multiple analytes at the point-of-care using a magneto-immunoassay in lateral flow format.

The dual role of paramagnetic particles for integrated lysis and measurement in a rapid immunoassay for intracellular proteins.

A novel, integrated lysis and immunoassay methodology and system for intracellular protein measurement are described. The method uses paramagnetic particles both as a lysis agent and assay label resulting in a rapid test requiring minimal operator intervention, the test being homogeneous and completed in less than 10 min. A design study highlights the critical features of the magnetic detection system used to quantify the paramagnetic particles and a novel frequency-locked loop-based magnetometer is presented. A study of paramagnetic particle enhanced lysis demonstrates that the technique is more than twice as efficient at releasing intracellular protein as ultrasonic lysis alone. Results are presented for measurements of intracellular prostate specific antigen in an LNCAP cell line. This model was selected to demonstrate the rapidity and efficiency of intracellular protein quantification. It was shown that, on average, LNCAP cells contained 0.43 fg of prostate specific antigen. This system promises an attractive solution for applications that require a rapid determination of intracellular proteins.

Novel immunoassay technique for rapid measurement of intracellular proteins using paramagnetic particles.

Magnetic immunoassays have been shown to have similar detection limits to conventional immunoassays, with the advantage of reduced total assay time. The aim of this study was to demonstrate that an integrated lysis and measurement system could be used to quantitatively measure intracellular target molecules using prostate specific antigen as a model analyte. The system described utilises the inherent physical properties of paramagnetic particles for both cell lysis and antigen quantification in the same vessel. This is achieved using ultrasound to energise paramagnetic particles to lyse cells combined with a magnetic immunoassay to measure intracellular protein, synthesised within the cells. Antibody coated paramagnetic particles were energised using pulses of ultrasound energy to penetrate and lyse cells and capture intracellular protein on the particle surface. The particles were drawn to an antibody coated sensor surface, in an applied magnetic field, which bound to captured analyte on the paramagnetic particle. The number of immobilised particles on the sensor surface is quantified by a resonant coil magnetometer. The total assay time was reduced to less than 15min. To demonstrate the utility of the system a model assay for intracellular prostate specific antigen was developed to show that the assay could detect differences in the amount of intracellular protein. This was achieved by exposing LNCaP cells to increasing concentrations of testosterone, which causes an increase in prostate specific antigen production. The rapid intracellular assay was able to demonstrate increasing amounts of intracellular prostate specific antigen resulting from increasing testosterone exposure. The technology could be used to develop rapid diagnostic tests for intracellular biomarkers that are difficult to detect in normal serum samples, for example viral proteins and intracellular cancer proteins.

Use of external magnetic fields to reduce reaction times in an immunoassay using micrometer-sized paramagnetic particles as labels (magnetoimmunoassay).

The paper presents a rapid immunoassay system capable of quantifying analyte in complex biological and environmental media. Antibody-coated micrometer-sized paramagnetic particles are used as labels in an assay in which they bind quantitatively with an analyte and capture antibody molecules immobilized on a polyester disk to form a sandwich assay. The assay is performed in a simple reaction vessel, and reactions between labels, analyte, and antibodies are accelerated by positioning magnets alternately above and below the vessel. The bound paramagnetic particles are quantified using a simple flat, spiral, coil located just below the polyester disk. The electronic circuitry associated with the coil uses components that are inexpensive and readily available. The coil has been designed to respond only to particles bound on the disk and not to unbound particles still in the test solution. Unbound particles are pulled away from the disk by the magnet before readings are taken. The use of the reaction vessel with the cardiac markers CRP and CKMB is described. No sample preparation or washing step is used in the assays, and results can be obtained in less than 3 min after introducing the sample into the vessel with sensitivities in the normal clinical range.