Jaime Castillo-León

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.

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.

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.

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.

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.

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.

Alignment and Use of Self-Assembled Peptide Nanotubes as Dry-Etching Mask

Self-assembled diphenylalanine peptide nanotubes provide a means of achieving nanostructured materials in a very simple and fast way. Recent discoveries have shown that this unique material, in addition to remaining stable under dry conditions, rapidly dissolves in water making it a promising candidate for controlled nanofabrication without organic solvents. The present work demonstrates how this unique structure can be aligned, manipulated and used as both an etching mask in a dry etching procedure and as a lift-off material. As a further demonstration of the potential of this technique, the peptide nanotubes were utilized to fabricate silicon nanowire devices and gold nanoslits in a rapid manner.

Self–Assembled Peptide Nanostructures for Biomedical Applications: Advantages and Challenges

Over the last 20 years, self-assembled nanostructures based on peptides have been investigated and presented as biomaterials with an impressive potential to be used in different bionanotechnological applications such as sensors, drug delivery systems, bioelectronics, tissue reparation, among others. Several advantages (mild synthesis conditions, relatively simple functionalization, low-cost and fast synthesis) confirm the promise of these biological nanostructures as excellent candidates for such uses. Through self-assembly, peptides can give rise to a range of well-defined nanostructures such as nanotubes, nanofibers, nanoparticles, nanotapes, gels and nanorods. However, there are several challenges that have yet to be extensively approached and solved. Issues like controlling the size during synthesis, the stability in liquid environments and manipulation have to be confronted when trying to integrate these nanostructures in the development of sensing devices or drug-delivery systems. The fact that these issues present difficulties is reflected in the low number of devices or systems using this material in real applications. The present chapter discusses these challenges and presents possible solutions. A review of the state-of-the-art work concerning the use of peptide self-assembled structures in biomedical applications is given. Additionally, our findings regarding the on-chip synthesis of peptide self-assembled nanotubes and nanoparticles, their controlled manipulation, as well as electrical and structural characterizations are introduced. Our latest results showing the interaction of peptide self-assembled structures with cells for the development of a combined sensing/cell culture platform and the use of these material in clean-room processes together with the stability of the biological structures in liquid are also presented.

Microfluidics and Lab-on-a-Chip Devices: History and Challenges

The rapid advances in microfabrication and nanofabrication in combination with the synthesis and discovery of new materials have propelled the drive to develop new technological devices such as smartphones, personal and tablet computers. These devices have changed the way humankind interacts and communicates and this change occurred very quickly due in part to decreased production and commercialization costs. As a result, not only nations with powerful economies but also emerging economies and poor countries can get access to these technologies and experience new ways to interact and instantly communicate what is happening around us. Following the advances of all these communication devices as well as those in microfabrication and nanofabrication and the emergence of new materials, technologies such as lab-on-a-chip (LOC) and micro total analysis systems (microTAS) were also boosted, albeit at a slower pace. LOC and microTAS applications have principally been utilized in the biomedical, food and environmental fields. But lately they have also found their place in the synthesis of new chemical compounds and the fabrication of nanostructures. It has become obvious that the LOC and microTAS technologies need to join forces with those behind the new communication devices which provide sources of power, detection and data transmission complementing the features that lab-on-a-chip and microTAS platforms can offer. An increasing number of microfluidic-based devices, developed both in small start-ups and large pharmaceutical and biomedical companies, is being released and entering the market. This chapter offers an overview of the first events in the history of LOC and microTAS devices, the biggest achievements and the challenges that still need to be overcome in order to accelerate the use of this technology.

Fabrication of Nanostructures Using Self-Assembled Peptides as Templates: The Diphenylalanine Case

This chapter evaluates the use of a short-aromatic dipeptide, diphenylalanine, as a template in the fabrication of new nanostructures (nanowires, coaxial nanocables, nanochannels) using materials such as silicon, conducting and non-conducting polymers. Diphenylalanine self-organize into nanostructures (nanotubes, nanofibers or nanospheres) under very mild conditions; some of its properties make them excellent candidates to be use as, for instance, dry-etch masks in a reactive ion etching process for the rapid fabrication of silicon micro and nanowires. Here, the methods used to exploit the advantages of diphenylalanine are explained step by step offering new alternatives to fabricate nanostructures in a simple and rapid way. The chapter is complemented with techniques to manipulate the self-assembled diphenylalanine nanostructures without changing its properties during the manipulation process.