Commercially available rapid diagnostic tests for the detection of high priority pathogens: status and challenges.

The ongoing COVID-19 pandemic has shown the importance of having analytical devices that allow a simple, fast, and robust detection of pathogens which cause epidemics and pandemics. The information these devices can collect is crucial for health authorities to make effective decisions to contain the disease's advance. The World Health Organization published a list of primary pathogens that have raised concern as potential causes of future pandemics. Unfortunately, there are no rapid diagnostic tests commercially available and approved by the regulatory bodies to detect most of the pathogens listed by the WHO. This report describes these pathogens, the available detection methods, and highlights areas where more attention is needed to produce rapid diagnostic tests for future pandemic surveillance.

Doped overoxidized polypyrrole microelectrodes as sensors for the detection of dopamine released from cell populations.

A surface modification of interdigitated gold microelectrodes (IDEs) with a doped polypyrrole (PPy) film for detection of dopamine released from populations of differentiated PC12 cells is presented. A thin PPy layer was potentiostatically electropolymerized from an aqueous pyrrole solution onto electrode surfaces. The conducting polymer film was doped during electropolymerization by introducing counter-ions in the monomer solution. Several counter-ions were tested and the resulting electrode modifications were characterized electrochemically to find the optimal dopant that increases sensitivity in dopamine detection. Overoxidation of the PPy films was shown to contribute to a significant enhancement in sensitivity to dopamine. The changes caused by overoxidation in the electrochemical behavior and electrode morphology were investigated using cyclic voltammetry and SEM as well as AFM, respectively. The optimal dopant for dopamine detection was found to be polystyrene sulfonate anion (PSS(-)). Rat pheochromocytoma (PC12) cells, a suitable model to study exocytotic dopamine release, were differentiated on IDEs functionalized with an overoxidized PSS(-)-doped PPy film. The modified electrodes were used to amperometrically detect dopamine released by populations of cells upon triggering cellular exocytosis with an elevated K(+) concentration. A comparison between the generated current on bare gold electrodes and gold electrodes modified with overoxidized doped PPy illustrates the clear advantage of the modification, yielding 2.6-fold signal amplification. The results also illustrate how to use cell population based dopamine exocytosis measurements to obtain biologically significant information that can be relevant in, for instance, the study of neural stem cell differentiation into dopaminergic neurons.

Combined cell culture-biosensing platform using vertically aligned patterned peptide nanofibers for cellular studies.

This Article presents the development of a combined cell culture-biosensing platform using vertically aligned self-assembled peptide nanofibers. Peptide nanofibers were patterned on a microchip containing gold microelectrodes to provide the cells with a 3D environment enabling them to grow and proliferate. Gold microelectrodes were functionalized with conductive polymers for the electrochemical detection of dopamine released from PC12 cells. The combined cell culture-biosensing platform assured a close proximity of the release site, the cells and the active surface of the sensor, thereby rendering it possible to avoid a loss of sensitivity because of the diffusion of the sample. The obtained results showed that the peptide nanofibers were suitable as a cell culturing substrate for PC12 cells. The peptide nanofibers could be employed as an alternative biological material to increase the adherence properties of PC12 cells. Dopamine was amperometrically detected at a value of 168 fmole.

Dielectrophoretic manipulation and solubility of protein nanofibrils formed from crude crystallins.

Protein nanofibrils and nanotubes are now widely accepted as having potential for use in the field of bionanotechnology. For this to be a feasible alternative to existing technologies, there is a need for a commercially viable source. Previous work has identified amyloid fibrils formed from crude crystallin proteins as such a source, since these fibrils can be produced in large quantities at a low cost. Applications include use of fibrils as templates for the formation of nanowires or as biosensing scaffolds. There remains a number of practical considerations, such as stability and the ability to control their arrangement. In this study, crude crystallin amyloid fibrils are shown to be stable in a range of biological and clean room solvents, with the fibril presence confirmed by transmission electron microscopy and the thioflavin T fluorescent assay. The fibrils were also immobilised between microelectrodes using dielectrophoresis, which enabled the recording of I-V curves for small numbers of fibrils. This investigation showed the fibrils to have low conductivity, with current values in the range of 10(-10) A recorded. This low conductivity could be increased through modification, or alternately, the fibrils could be used unmodified for applications where they can act as templates or high surface area nanoscaffolds.

Detection of cancer cells using a peptide nanotube-folic acid modified graphene electrode.

This article describes the preparation of a graphene electrode modified with a new conjugate of peptide nanotubes and folic acid for the selective detection of human cervical cancer cells over-expressing folate receptors. The functionalization of peptide nanotubes with folic acid was confirmed by fluorescence microscopy and atomic force microscopy. The peptide nanotube-folic acid modified graphene electrode was characterized by scanning electron microscopy and cyclic voltammetry. The modification of the graphene electrode with peptide nanotube-folic acid led to an increase in the current signal. The human cervical cancer cells were bound to the modified electrode through the folic acid-folate receptor interaction. Cyclic voltammograms in the presence of [Fe(CN)(6)](3-/4-) as a redox species demonstrated that the binding of the folate receptor from human cervical cancer cells to the peptide nanotube-folic acid modified electrode lowered the electron transfer resulting in a decrease in the measured current. A detection limit of 250 human cervical cancer cells per mL was obtained. Control experiments confirmed that the peptide nanotube-folic acid electrode specifically recognized folate receptors. The modified electrode described here opens up new possibilities for future applications in early stage diagnoses of diseases where cells over-express folate receptors, such as in cancer or leishmaniasis disease.

Non-covalent conjugates of single-walled carbon nanotubes and folic acid for interaction with cells over-expressing folate receptors.

We here present a method to form a noncovalent conjugate of single-walled carbon nanotubes and folic acid aimed to interact with cells over-expressing folate receptors. The bonding was obtained without covalent chemical functionalization using a simple, rapid "one pot" synthesis method. The zeta potential for the single-walled carbon nanotube-folic acid solution was -32.4 mV at pH 7.0 and the result indicates that the folic acid coating inhibited aggregation of the carbon nanotubes. Properties of the single-walled carbon nanotube-folic acid conjugate were analyzed using ultraviolet-visible, fluorescence and Raman spectroscopies. While the folic acid fluorescence signature was significantly quenched by the presence of single-walled carbon nanotubes, the Raman spectra of the conjugate displayed a decreased distribution of sp3 sites. Both results were attributed to the noncovalent functionalization of the single-walled carbon nanotubes with folic acid. A more detailed investigation of the single-walled carbon nanotube-folic acid conjugates utilizing scanning electron microscopy, atomic force microscopy and energy-dispersive X-ray spectroscopy confirmed the presence of the well-defined folic acid coating on the individual single-walled carbon nanotubes. The single-walled carbon nanotube-folic acid conjugates were incubated with THP-1 cells and the internalization was evaluated by Giemsa staining with light microscopy, and cytotoxicity was evaluated using the MTT reduction assay. The cytotoxicity studies presented a low toxicity of the conjugates in the THP-1 cells. The low toxicity and the cellular uptake of single-walled carbon nanotube-folic acid by cancer cells suggest their potential use in carbon nanotube-based drug delivery systems and in the diagnosis of cancer or tropical diseases such as leishmaniasis.

Self-assembled diphenylalanine nanowires for cellular studies and sensor applications.

In this paper we present a series of experiments showing that vertical self-assembled diphenylalanine peptide nanowires (PNWs) are a suitable candidate material for cellular biosensing. We grew HeLa and PC12 cells onto PNW modified gold surfaces and observed no hindrance of cell growth caused by the peptide nanostructures; furthermore we studied the properties of PNWs by investigating their influence on the electrochemical behavior of gold electrodes. The PNWs were functionalized with polypyrrole (PPy) by chemical polymerization, therefore creating conducting peptide/polymer nanowire structures vertically attached to a metal electrode. The electroactivity of such structures was characterized by cyclic voltammetry. The PNW/PPy modified electrodes were finally used as amperometric dopamine sensors, yielding a detection limit of 3,1 microM.

Electrostatic force microscopy of self-assembled peptide structures.

In this report electrostatic force microscopy (EFM) is used to study different peptide self-assembled structures such as tubes and particles. It is shown that not only geometrical information can be obtained using EFM, but also information about the composition of different structures. In particular we use EFM to investigate the structures of diphenylalanine peptide tubes, particles, and CSGAITIG peptide particles placed on pre-fabricated SiO(2) surfaces with a backgate. We show that the cavity in the peptide tubes could be due to the presence of water residues. Additionally we show that self-assembled amyloid peptides form spherical solid structures containing the same self-assembled peptide in its interior. In both cases transmission electron microscopy is used to verify these structures. Further, the limitations of the EFM technique are discussed, especially when the observed structures become small compared with the radius of the AFM tip used. Finally, an agreement between the detected signal and the structure of the hollow peptide tubes is demonstrated.

Development of an electrochemical metal-ion biosensor using self-assembled peptide nanofibrils.

This article describes the combination of self-assembled peptide nanofibrils with metal electrodes for the development of an electrochemical metal-ion biosensor. The biological nanofibrils were immobilized on gold electrodes and used as biorecognition elements for the complexation with copper ions. These nanofibrils were obtained under aqueous conditions, at room temperature and outside the clean room. The functionalized gold electrode was evaluated by cyclic voltammetry, impedance spectroscopy, energy dispersive X-ray and atomic force microscopy. The obtained results displayed a layer of nanofibrils able to complex with copper ions in solution. The response of the obtained biosensor was linear up to 50 µM copper and presented a sensitivity of 0.68 µA cm⁻² µM⁻¹. Moreover, the fabricated sensor could be regenerated to a copper-free state allowing its reutilization.

Stability of diphenylalanine peptide nanotubes in solution.

Over the last couple of years, self-organizing nanotubes based on the dipeptide diphenylalanine have received much attention, mainly as possible building blocks for the next generation of biosensors and as drug delivery systems. One of the main reasons for this large interest is that these peptide nanotubes are believed to be very stable both thermally and chemically. Previously, the chemical and thermal stability of self-organizing structures has been investigated after the evaporation of the solvent. However, it was recently discovered that the stability of the structures differed significantly when the tubes were in solution. It has been shown that, in solution, the peptide nanotubes can easily be dissolved in several solvents including water. It is therefore of critical importance that the stability of the nanotubes in solution and not after solvent evaporation be investigated prior to applications in which the nanotube will be submerged in liquid. The present article reports results demonstrating the instability and suggests a possible approach to a stabilization procedure, which drastically improves the stability of the formed structures. The results presented herein provide new information regarding the stability of self-organizing diphenylalanine nanotubes in solution.

Conducting polymer 3D microelectrodes.

Conducting polymer 3D microelectrodes have been fabricated for possible future neurological applications. A combination of micro-fabrication techniques and chemical polymerization methods has been used to create pillar electrodes in polyaniline and polypyrrole. The thin polymer films obtained showed uniformity and good adhesion to both horizontal and vertical surfaces. Electrodes in combination with metal/conducting polymer materials have been characterized by cyclic voltammetry and the presence of the conducting polymer film has shown to increase the electrochemical activity when compared with electrodes coated with only metal. An electrochemical characterization of gold/polypyrrole electrodes showed exceptional electrochemical behavior and activity. PC12 cells were finally cultured on the investigated materials as a preliminary biocompatibility assessment. These results show that the described electrodes are possibly suitable for future in-vitro neurological measurements.