Jonathan M. Cooper

A review of the immobilization of enzymes in electropolymerized films

Abstract The use of electrochemically polymerized films to immobilize enzymes at electrode surfaces is reviewed, and the interpretation and modelling of the results from these studies is discussed.

Microfluidic Single-Cell Array Cytometry for the Analysis of Tumor Apoptosis

Limitations imposed by conventional analytical technologies for cell biology, such as flow cytometry or microplate imaging, are often prohibitive for the kinetic analysis of single-cell responses to therapeutic compounds. In this paper, we describe the application of a microfluidic array to the real-time screening of anticancer drugs against arrays of single cells. The microfluidic platform comprises an array of micromechanical traps, designed to passively corral individual nonadherent cells. This platform, fabricated in the biologically compatible elastomer poly(dimethylsiloxane), PDMS, enables hydrodynamic trapping of cells in low shear stress zones, enabling time-lapse studies of nonadherent hematopoietic cells. Results indicate that these live-cell, microfluidic microarrays can be readily applied to kinetic analysis of investigational anticancer agents in hematopoietic cancer cells, providing new opportunities for automated microarray cytometry and higher-throughput screening. We also demonstrate the ability to quantify on-chip the anticancer drug induced apoptosis. Specifically, we show that with small numbers of trapped cells (approximately 300) under careful serial observation we can achieve results with only slightly greater statistical spread than can be obtained with single-pass flow cytometer measurements of 15,000-30,000 cells.

Tumors on chips: oncology meets microfluidics

Despite over 2 million papers published on cancer so far, malignancy still remains a puzzlingly complex disease with overall low survival rates. Expanding our knowledge of the molecular mechanisms of malignancy and of resistance to therapy is crucial in guiding the successful design of anti-cancer drugs and new point-of-care diagnostics. The up-and-coming microfluidic Lab-on-a-Chip (LOC) technology and micro-total analysis systems (μTAS) are arguably the most promising platforms to address the inherent complexity of cellular systems with massive experimental parallelization and 4D analysis on a single cell level. This review discusses the emerging applications of microfluidic technologies and their advantages for cancer biology and experimental oncology. We also summarize the recent advances in miniaturized systems to study cancer cell microenvironment, cancer cytomics, and real-time (4D) pharmacological screening. Microfabricated systems, such as cell microarrays, together with on-chip label-less cytometry, and micro-sorting technologies, are all highlighted with the view of describing their potential applications in pharmacological screening, drug discovery, and clinical oncology. It is envisaged that microfluidic solutions may well represent the platform of choice for next generation in vitro cancer models.

Microfluidic single cell arrays to interrogate signalling dynamics of individual, patient-derived hematopoietic stem cells

Stem cells hold great promise as a means of treating otherwise incurable, degenerative diseases due to their ability both to self-renew and differentiate. However, stem cell damage can also play a role in the disease with the formation of solid tumors and leukaemias such as chronic myeloid leukaemia (CML), a hematopoietic stem cell (HSC) disorder. Despite recent medical advances, CML remains incurable by drug therapy. Understanding the mechanisms which govern chemoresistance of individual stem cell leukaemias may therefore require analysis at the single cell level. This task is not trivial using current technologies given that isolating HSCs is difficult, expensive, and inefficient due to low cell yield from patients. In addition, hematopoietic cells are largely non-adherent and thus difficult to study over time using conventional cell culture techniques. Hence, there is a need for new microfluidic platforms that allow the functional interrogation of hundreds of non-adherent single cells in parallel. We demonstrate the ability to perform assays, normally performed on the macroscopic scale, within the microfluidic platform using minimal reagents and low numbers of primary cells. We investigated normal and CML stem cell responses to the tyrosine kinase inhibitor, dasatinib, a drug approved for the treatment of CML. Dynamic, on-chip three-color cell viability assays revealed that differences in the responses of normal and CML stem/progenitor cells to dasatinib were observed even in the early phases of exposure, during which time normal cells exhibit a significantly elevated cell death rate, as compared to both controls and CML cells. Further studies show that dasatinib does, however, markedly reduce CML stem/progenitor cell migration in situ.

Direct electron transfer reactions between immobilized cytochrome c and modified gold electrodes

In this paper we present data showing quasi-reversible electrochemistry of soluble cytochrome c using the gold electrode modifier N-acetyl cysteine (E12=25 MV, ΔEp=60 mV, Ip,a/ν12=constant). Cytochrome c was subsequently immobilized at this modified electrode using a carbodimide condensation reaction. The electron-transfer rate between the immobilized protein and the gold electrode was estimated as 3.4 ± 1.2 s−1 and the formal potential E°′ of the immobilized protein electrode was calculated as 2 mV/SCE. We also present data showing an application for the immobilized cytochrome c electrode as a sensor for the specific measurement of superoxide radical production.

Protein Expression, Aggregation, and Triggered Release from Polymersomes as Artificial Cell‐like Structures

Bringing droplets to life: A cytoskeletal protein (red dots, see scheme) is expressed in artificial cells composed of biocompatible polymersomes, which encapsulate expression machinery and amino acid building blocks. Release of the expressed proteins can be triggered by a negative osmotic shock.

Surface Acoustic Wave Nebulization of Peptides as a Microfluidic Interface for Mass Spectrometry

We describe the fabrication of a surface acoustic wave (SAW) device on a LiNbO(3) piezoelectric transducer for the transfer of nonvolatile analytes to the gas phase at atmospheric pressure (a process referred to as nebulization or atomization). We subsequently show how such a device can be used in the field of mass spectrometry (MS) detection, demonstrating that SAW nebulization (SAWN) can be performed either in a discontinuous or pulsed mode, similar to that for matrix assisted laser desorption ionization (MALDI) or in a continuous mode like electrospray ionization (ESI). We present data showing the transfer of peptides to the gas phase, where ions are detected by MS. These peptide ions were subsequently fragmented by collision-induced dissociation, from which the sequence was assigned. Unlike MALDI mass spectra, which are typically contaminated with matrix ions at low m/z, the SAWN generated spectra had no such interference. In continuous mode, the SAWN plume was sampled on a microsecond time scale by a linear ion trap mass spectrometer and produced multiply charged peptide precursor ions with a charge state distribution shifted to higher m/z compared to an identical sample analyzed by ESI. The SAWN technology also provides the opportunity to re-examine a sample from a flat surface, repeatedly. The process can be performed without the need for capillaries, which can clog, reservoirs, which dilute the sample, and electrodes, which when in direct contact with sample, cause unwanted electrochemical oxidation. In both continuous and pulsed sampling modes, the quality of precursor ion scans and tandem mass spectra of peptides was consistent across the plumes lifetime.

Microrheology with optical tweezers

Microrheology is the study of the flow of materials over small scales. It is of particular interest to those involved with investigations of fluid properties within Lab-on-a-Chip structures or within other micron-scale environments. The article briefly reviews existing active and passive methods used in the study of fluids. It then explores in greater detail the use of optical tweezers as an emerging method to investigate rheological phenomena, including, for example, viscosity and viscoelasticity, as well as the related topic of flow. The article also describes, briefly, potential future applications of this topic, in the fields of biological measurement, in general, and Lab-on-a-Chip, in particular.

Shaping acoustic fields as a toolset for microfluidic manipulations in diagnostic technologies

Ultrasonics offers the possibility of developing sophisticated fluid manipulation tools in lab-on-a-chip technologies. Here we demonstrate the ability to shape ultrasonic fields by using phononic lattices, patterned on a disposable chip, to carry out the complex sequence of fluidic manipulations required to detect the rodent malaria parasite Plasmodium berghei in blood. To illustrate the different tools that are available to us, we used acoustic fields to produce the required rotational vortices that mechanically lyse both the red blood cells and the parasitic cells present in a drop of blood. This procedure was followed by the amplification of parasitic genomic sequences using different acoustic fields and frequencies to heat the sample and perform a real-time PCR amplification. The system does not require the use of lytic reagents nor enrichment steps, making it suitable for further integration into lab-on-a-chip point-of-care devices. This acoustic sample preparation and PCR enables us to detect ca. 30 parasites in a microliter-sized blood sample, which is the same order of magnitude in sensitivity as lab-based PCR tests. Unlike other lab-on-a-chip methods, where the sample moves through channels, here we use our ability to shape the acoustic fields in a frequency-dependent manner to provide different analytical functions. The methods also provide a clear route toward the integration of PCR to detect pathogens in a single handheld system.

Toward a miniature wireless integrated multisensor microsystem for industrial and biomedical applications

This paper presents our work toward the integration of a multisensor microsystem with wireless communication, using system-on-chip (SoC) methodology. Four different forms of microelectronic sensors have been fabricated on two separate 5/spl times/5 mm/sup 2/ silicon chips measuring pH, conductivity, dissolved oxygen concentration, and temperature. The sensors are integrated with a sensor fusion chip comprising analog circuitry for sensor operation and signal amplification prior to digital decoding and transmission. The microsystem prototype will be packaged in a miniature capsule, which measures 16 mm /spl times/55 mm including batteries and dissipates 6.3 mW for a minimal life cycle of 12 h.