Gerardo Perozziello

Microchemostat—microbial continuous culture in a polymer-based, instrumented microbioreactor

In a chemostat, microbial cells reach a steady state condition at which cell biomass production, substrates and the product concentrations remain constant. These features make continuous culture a unique and powerful tool for biological and physiological research. We present a polymer-based microbioreactor system integrated with optical density (OD), pH, and dissolved oxygen (DO) real-time measurements for continuous cultivation of microbial cells. Escherichia coli (E. coli) cells are continuously cultured in a 150 microL, membrane-aerated, well-mixed microbioreactor fed by a pressure-driven flow of fresh medium through a microchannel. Chemotaxisial back growth of bacterial cells into the medium feed channel is prevented by local heating. Using poly(ethylene glycol) (PEG)-grafted poly(acrylic acid) (PAA) copolymer films, the inner surfaces of poly(methyl methacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS) of the microbioreactor are modified to generate bio-inert surfaces resistant to non-specific protein adsorption and cell adhesion. The modified surfaces of microbioreactor effectively reduce wall growth of E. coli for a prolonged period of cultivation. Steady state conditions at different dilution rates are demonstrated and characterized by steady OD, pH, and DO levels.

Superhydrophobic Surfaces as Smart Platforms for the Analysis of Diluted Biological Solutions

The aim of this paper is to expound on the rational design, fabrication and development of superhydrophobic surfaces (SHSs) for the manipulation and analysis of diluted biological solutions. SHSs typically feature a periodic array or pattern of micropillars; here, those pillars were modified to incorporate on the head, at the smallest scales, silver nanoparticles aggregates. These metal nanoclusters guarantee superior optical properties and especially SERS (surface enhanced Raman scattering) effects, whereby a molecule, adsorbed on the surface, would reveal an increased spectroscopy signal. On account of their two scale-hybrid nature, these systems are capable of multiple functions which are (i) to concentrate a solution, (ii) to vehicle the analytes of interest to the active areas of the substrate and, therefore, (iii) to measure the analytes with exceptional sensitivity and very low detection limits. Forasmuch, combining different technologies, these devices would augment the performance of conventional SERS substrates and would offer the possibility of revealing a single molecule. In this work, similar SHSs were used to detect Rhodamine molecules in the fairly low atto molar range. The major application of this novel family of devices would be the early detection of tumors or other important pathologies, with incredible advances in medicine.

Integrated microfluidic device for single-cell trapping and spectroscopy

Optofluidic microsystems are key components towards lab-on-a-chip devices for manipulation and analysis of biological specimens. In particular, the integration of optical tweezers (OT) in these devices allows stable sample trapping, while making available mechanical, chemical and spectroscopic analyses.

Microbioreactors for Bioprocess Development

As a step toward high-throughput bioprocess development, we present design, fabrication, and characterization of polymer based microbioreactors integrated with automated sensors and actuators. The devices are realized, in increasing levels of complexity, in poly(dimethylsiloxane) and poly(methyl methacrylate) by micromachining and multilayer thermal compression bonding procedures. Online optical measurements for optical density, pH, and dissolved oxygen are integrated. Active mixing is made possible by a miniature magnetic stir bar. Plug-in-and-flow microfluidic connectors and fabricated polymer micro-optical lenses/connectors are integrated in the microbioreactors for fast set up and easy operation. Application examples demonstrate the feasibility of culturing microbial cells, specifically Escherichia coli, in 150 μL-volume bioreactors in batch, continuous, and fed-batch operations. (JALA 2007;12:143–51)

Detection of single amino acid mutation in human breast cancer by disordered plasmonic self-similar chain

Novel nanoarray for single molecule detection from peptide mixture. Control of the architecture and electromagnetic behavior of nanostructures offers the possibility of designing and fabricating sensors that, owing to their intrinsic behavior, provide solutions to new problems in various fields. We show detection of peptides in multicomponent mixtures derived from human samples for early diagnosis of breast cancer. The architecture of sensors is based on a matrix array where pixels constitute a plasmonic device showing a strong electric field enhancement localized in an area of a few square nanometers. The method allows detection of single point mutations in peptides composing the BRCA1 protein. The sensitivity demonstrated falls in the picomolar (10−12 M) range. The success of this approach is a result of accurate design and fabrication control. The residual roughness introduced by fabrication was taken into account in optical modeling and was a further contributing factor in plasmon localization, increasing the sensitivity and selectivity of the sensors. This methodology developed for breast cancer detection can be considered a general strategy that is applicable to various pathologies and other chemical analytical cases where complex mixtures have to be resolved in their constitutive components.

Rapid prototyping tools and methods for all-Topas® cyclic olefin copolymer fluidic microsystems

Abstract Topas®, the cyclic olefin copolymer, from Topas Advanced Polymers GmbH has a number of advantages over polymers such as poly(methylmethacrylate), polydimethylsiloxane, and polycarbonate traditionally used in fluid microsystem manufacturing, such as low water absorption, high chemical resistance, good machinability, and good optical properties. A number of different processes for rapid and low-cost prototyping of all-Topas microfluidic systems, made with desktop machinery, are presented. Among the processes are micromilling of fluidic structures with a width down to 25 μm and sealing of fluidic channels by thermal bonding or laser bonding, using a thin, spin-coated layer of carbon particles between the Topas substrate and the lid to absorb the laser light.

Electroless Deposition and Nanolithography Can Control the Formation of Materials at the Nano-Scale for Plasmonic Applications

The new revolution in materials science is being driven by our ability to manipulate matter at the molecular level to create structures with novel functions and properties. The aim of this paper is to explore new strategies to obtain plasmonic metal nanostructures through the combination of a top down method, that is electron beam lithography, and a bottom up technique, that is the chemical electroless deposition. This technique allows a tight control over the shape and size of bi- and three-dimensional metal patterns at the nano scale. The resulting nanostructures can be used as constituents of Surface Enhanced Raman Spectroscopy (SERS) substrates, where the electromagnetic field is strongly amplified. Our results indicate that, in electroless growth, high quality metal nanostructures with sizes below 50 nm may be easily obtained. These findings were explained within the framework of a diffusion limited aggregation (DLA) model, that is a simulation model that makes it possible to decipher, at an atomic level, the rules governing the evolution of the growth front; moreover, we give a description of the physical mechanisms of growth at a basic level. In the discussion, we show how these findings can be utilized to fabricate dimers of silver nanospheres where the size and shape of those spheres is controlled with extreme precision and can be used for very large area SERS substrates and nano-optics, for single molecule detection.

A fast and reliable way to establish fluidic connections to planar microchips

In this work, we present a non-permanent method to connect microfluidic devices. The approach uses short flexible tubes that are plugged into bottom-flat holes and ensure fast and reliable interconnections. The small available dimensions allow the tube to be directly attached to the side of planar microchips. A theoretical model to estimate the maximum applicable pressure was developed, and verified with experimental data. Furthermore, the tube connections were compared to other non-permanent interconnection types.

A facile in situ microfluidic method for creating multivalent surfaces: toward functional glycomics

An in situ method of modifying the chemistry and topology of microfluidic surfaces in order to mimic the cellular environment is described. The binding of functionalised microbeads to microfluidic channels allows the surface-to-volume ratio of the system, and thus the number of biomolecules available for reaction, to be vastly increased, thereby enhancing the sensitivity of biochemical analyses. The sensitivity and specificity of the technique were first investigated via the study of carbohydrate-protein interactions. Beads featuring hydrazide moieties were adhered to the channel surface, after which carbohydrates (galactose and mannose) were bound to the beads in situ and reacted with fluorescently labelled proteins. Results showed a six-fold increase in fluorescent signal compared to the same process performed on a glass surface without the presence of beads, thereby demonstrating the increase in valence afforded by the method. In a subsequent study, beads, modified with galactose moieties via the in situ functionalisation technique, were used to perform studies of colon tumour cells from a cell sample. Here, the carcinoma cells exhibited superior adhesion than the normal cells due to an increased expression of active galactose receptors, thereby demonstrating the success of the biofunctionalisation method for investigating cellular mechanisms.

Protein–Carbohydrate Complex Reveals Circulating Metastatic Cells in a Microfluidic Assay

Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3--a protein that is correlated to the progress of tumor and metastasis--is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interactions strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine.