Integration of low-power microfluidic pumps with biosensors within a laboratory-on-a-chip device.

We describe the fabrication of a controllable microfluidic valve coupled with an electrochemical pump, which has been designed to deliver reagents to an integrated microfluidic biosensing system. Fluid, retained within an insertion reservoir using a stop valve, was pumped using electrochemical actuation, providing a low power, low voltage integrated Laboratory-on-a-Chip for reproducible, small volume fluidic manipulation. The properties of the valve were characterized using both X-ray photoelectron spectroscopy and contact angle measurements, enabling the calculation of the magnitude of the forces involved (which were subsequently verified through experimental measurement). Electrochemical generation of oxygen and hydrogen acted as an on-demand pressure system to force fluid over the stop valve barrier. The process of filling-up the biosensing chamber was characterized in terms of the time to fill, the energy used, and the peak power consumed. The potential of the device was illustrated using a glucose biosensor.

Characterization of cellular chemical dynamics using combined microfluidic and Raman techniques.

The integration of a range of technologies including microfluidics, surface-enhanced Raman scattering and confocal microspectroscopy has been successfully used to characterize in situ single living CHO (Chinese hamster ovary) cells with a high degree of spatial (in three dimensions) and temporal (1 s per spectrum) resolution. Following the introduction of a continuous flow of ionomycin, the real time spectral response from the cell was monitored during the agonist-evoked Ca(2+) flux process. The methodology described has the potential to be used for the study of the cellular dynamics of a range of signalling processes.

Continuous flow separation of particles within an asymmetric microfluidic device.

A microfluidic based device has been developed for the continuous separation of polymer microspheres, taking advantage of the flow characteristics of systems. The chip consists of an asymmetric cavity with variable channel width which enables continuous amplification of the particle separation for different size particles within the laminar flow profile. The process has been examined by varying the sample inlet position, the sample to media flow rate ratio, and the total flow rate. This technique can be applied for manipulating both microscale biological and colloidal particles within microfluidic systems.

Implementation of radiotelemetry in a lab-in-a-pill format.

A miniaturised lab-in-a-pill device has been produced incorporating a temperature and pH sensor with wireless communication using the 433.92 MHz ISM band. The device has been designed in order to enable real time in situ measurements in the gastrointestinal (GI) tract, and accordingly, issues concerning the resolution and accuracy of the data, and the lifetime of the device have been considered. The sensors, which will measure two key parameters reflecting the physiological environment in the GI (as indicators for disease) were both controlled by an application specific integrated circuit (ASIC). The data were sampled at 10-bit resolution prior to communication off chip as a single interleaved data stream. This incorporated a power saving serial bitstream data compression algorithm that was found to extend the service lifetime of the pill by 70%. An integrated on-off keying (OOK) radio transmitter was used to send the signal to a local receiver (base station), prior to acquisition on a computer. A permanent magnet was also incorporated in the device to enable non-visual tracking of the system. We report on the implementation of this device, together with an initial study sampling from within the porcine GI tract, showing that measurements from the lab-on-a-pill, in situ, was within 90% of literature values.

Biocompatibility of a lab-on-a-pill sensor in artificial gastrointestinal environments.

In this paper, we present a radiotelemetry sensor, designed as a lab-in-a-pill, which incorporates a two-channel microfabricated sensor platform for real-time measurements of temperature and pH. These two parameters have potential application for use in remote biological sensing (for example they may be used as markers that reflect the physiological environment or as indicators for disease, within the gastrointestinal tract). We have investigated the effects of biofouling on these sensors, by exploring their response time and sensitivity in a model in vitro gastrointestinal system. The artificial gastric and intestinal solutions used represent a model both for fasting, as well as for the ingestion of food and subsequent digestion to gastrointestinal chyme. The results showed a decrease in pH sensitivity after exposure of the sensors for 3 h. The response time also increased from an initial measurement time of 10 s in pure GI juice, to ca. 25 s following the ingestion of food and 80 s in simulated chyme. These in vitro results indicate that changes in viscosity in our model gastrointestinal system had a pronounced effect on the unmodified sensor.

Evaluating the influence of deposition conditions on solvation of reactive conducting polymers with neutron reflectivity.

We describe in situ neutron reflectivity (NR) and RAIRS studies of the chemical modification of films of a polypyrrole-based conducting polymer derived from the pentafluorophenyl ester of poly(pyrrole-N-propanoic acid) (PFP) electrodeposited on electrode surfaces. We explore the role of the solvent in controlling the rate of reaction with solution-based nucleophiles (amines, which react with the ester to form amides). By varying the identity of the solvent (water vs acetonitrile) and the neutron contrast (deuteration), we find that both the identity of the solvent and its population within the film are paramount in determining chemical reactivity and electroactivity. IR signatures allow monitoring of the reaction of solution-based amine-tagged species such as amino-terminated poly(propylene glycol), ferrocene ethylamine, and lysine with film-based ester functionalities: the carbonyl bands show ester/amide interconversion and some hydrolysis to acid. Time-dependent spectral analysis shows marked variations in reaction rate with (i) (co-)polymer composition (replacement of some fluorinated ester-functionalized pyrrole with unfunctionalized pyrrole), (ii) the solvent to which the polymer film is exposed, and (iii) the rate of polymer deposition. NR data provide solvent profiles as a function of distance perpendicular to the interface, the variations of which provide an explanation for film reactivity patterns. Homopolymer films are relatively hydrophobic, thus hindering reaction with species present in water solutions. Incorporating pyrrole groups raises the solvent population-dramatically for water-thereby facilitating entry and reaction of aqueous-based lysine. Changing film deposition rate yields films with different absolute levels of solvent and reactivity patterns that are dependent on the size of the reactant molecules: more rapid deposition of polymer gives films with a more open structure leading to a higher solvent content and thence increased reactivity. These results, supported by XPS and AFM data, allow assembly of composition-structure-reactivity correlations, in which the controlling feature is film solvation.

A suspended membrane nanocalorimeter for ultralow volume bioanalysis.

A nanocalorimetric suspended membrane sensor for pL volumes of aqueous media was fabricated by bulk silicon micromachining using anisotropic wet etching and photo and electron beam lithographic techniques. A high-temperature sensitivity of 125 microK and a rapid unfiltered time constant of 12 ms have been achieved by integrating a miniaturized reaction vessel of 0.7-nL volume on a 800-nm-thick and 300 x 300- microm2-large silicon nitride membrane, thermally insulated from the surrounding bulk silicon. The combination of a ten-junction gold and nickel thermoelectric sensor with an integrated ultralow noise preamplifier has enabled the resolution of 15-nW power in a single measurement, a result confirmed by electrical calibration. The combination of a high sensitivity and rapid response time is a consequence of miniaturization. The choice of gold and nickel as sensor material provided the maximum thermal sensitivity in the context of ease of fabrication and cost. The nanocalorimetric sensor has the potential for integration in an ultralow-volume high-density array format for the characterization of processes in which there is an exchange of heat.

Generation of primary hepatocyte microarrays by piezoelectric printing.

We demonstrate a single-step method for the generation of collagen and poly-l-Lysine (PLL) micropatterns on a poly(ethylene glycol) (PEG) functionalized glass surface for cell based assays. The method involves establishing a reliable silanization method to create an effective non-adhesive PEG layer on glass that inhibits cell attachment, followed by the spotting of collagen or PLL solutions using non-contact piezoelectric printing. We show for the first time that the spotted protein micropatterns remain stable on the PEG surface even after extensive washing, thus significantly simplifying protein pattern formation. We found that adherence and spreading of NIH-3T3 fibroblasts was confined to PLL and collagen areas of the micropatterns. In contrast, primary rat hepatocytes adhered and spread only on collagen micropatterns, where they formed uniform, well defined functionally active cell arrays. The differing affinity of hepatocytes and NIH-3T3 fibroblasts for collagen and PLL patterns was used to develop a simple technique for creating a co-culture of the two cell types. This has the potential to form structured arrays that mimic the in vivo hepatic environment and is easily integrated within a miniaturized analytical platform for developing high throughput toxicity analysis in vitro.

Real time characterization of hydrodynamics in optically trapped networks of micro-particles.

The hydrodynamic interactions of micro-silica spheres trapped in a variety of networks using holographic optical tweezers are measured and characterized in terms of their predicted eigenmodes. The characteristic eigenmodes of the networks are distinguishable within 20-40 seconds of acquisition time. Three different multi-particle networks are considered; an eight-particle linear chain, a nine-particle square grid and, finally, an eight-particle ring. The eigenmodes and their decay rates are shown to behave as predicted by the Oseen tensor and the Langevin equation, respectively. Finally, we demonstrate the potential of using our micro-ring as a non-invasive sensor to the local environmental viscosity, by showing the distortion of the eigenmode spectrum due to the proximity of a planar boundary.

A microfluidic-based system for analysis of single cells based on Ca2+ flux.

A microfluidic format-based system has been developed for in situ monitoring of the calcium flux response to agonists using Chinese hamster ovary (CHO) cells. The assay is based on measuring the fluorescent intensity of the calcium-sensitive indicator, Fluo-4 AM, and was performed in a modified glass chip channel, whose surface was functionalised using a silanisation method with 3-aminopropyltriethoxysilane (APTS) (enabling the cells to be immobilised on the channel surface). CHO cells calcium flux response was measured for different agonists over a range of concentrations. Cells and reagents were introduced into the chip in a continuous flow as a series of plugs in a given sequence.

Analysis of protein adsorption and binding at biosensor polymer interfaces using X-ray photon spectroscopy and scanning electrochemical microscopy.

We describe a method, based on X-ray photoelectron spectroscopy (XPS) measurements, to assess the extent of protein adsorption or binding on a variety of different muTAS and biosensor interfaces. Underpinning this method is the labeling of protein molecules with either iodine- or bromine-containing motifs by using protocols previously developed for radiotracer studies. Using this method, we have examined the adsorption and binding properties of a variety of modified electrodeposited polymer interfaces as well as other materials used in muTAS device fabrication. Using polymer interfaces modified with poly(propylene glycol) (PPG) chains, our results indicate that a chain of at least approximately 30 monomer units is required to inhibit nonspecific adsorption from concentrated protein solutions. The XPS methodology was also used to probe specific binding of avidins and enzyme conjugates thereof to biotinylated and mixed biotin/PPG-modified polymer interfaces. In one example, using competitive binding, it was established that the mode of binding of a peroxidase-streptavidin conjugate to a biotinylated modified polymer interface was primarily via the streptavidin moiety (as opposed to nonspecific binding via the enzyme conjugate). XPS evaluation of nonspecific and specific peroxidase-streptavidin immobilization on various functionalized polymers has guided the design and fabrication of functionalized interdigitated electrodes in a biosensing muTAS device. Subsequent characterization of this device using scanning electrochemical microscopy (SECM) corroborated the adsorption and binding previously inferred from XPS measurements on macroscale electrodes.