High-resolution microcontact printing and transfer of massive arrays of microorganisms on planar and compartmentalized nanoporous aluminium oxide.

Handling microorganisms in high throughput and their deployment into miniaturized platforms presents significant challenges. Contact printing can be used to create dense arrays of viable microorganisms. Such "living arrays", potentially with multiple identical replicates, are useful in the selection of improved industrial microorganisms, screening antimicrobials, clinical diagnostics, strain storage, and for research into microbial genetics. A high throughput method to print microorganisms at high density was devised, employing a microscope and a stamp with a massive array of PDMS pins. Viable bacteria (Lactobacillus plantarum, Esherichia coli), yeast (Candida albicans) and fungal spores (Aspergillus fumigatus) were deposited onto porous aluminium oxide (PAO) using arrays of pins with areas from 5 x 5 to 20 x 20 microm. Printing onto PAO with up to 8100 pins of 20 x 20 microm area with 3 replicates was achieved. Printing with up to 200 pins onto PAO culture chips (divided into 40 x 40 microm culture areas) allowed inoculation followed by effective segregation of microcolonies during outgrowth. Additionally, it was possible to print mixtures of C. albicans and spores of A. fumigatus with a degree of selectivity by capture onto a chemically modified PAO surface. High resolution printing of microorganisms within segregated compartments and on functionalized PAO surfaces has significant advantages over what is possible on semi-solid surfaces such as agar.

On-chip determination of spermatozoa concentration using electrical impedance measurements.

In this article we describe the development of a microfluidic chip to determine the concentration of spermatozoa in semen, which is a main quality parameter for the fertility of a man. A microfluidic glass-glass chip is used, consisting of a microchannel with a planar electrode pair that allows the detection of spermatozoa passing the electrodes using electrical impedance measurements. Cells other than spermatozoa in semen also cause a change in impedance when passing the electrodes, interfering with the spermatozoa count. We demonstrate that the change in electrical impedance is related to the size of cells passing the electrodes, allowing to distinguish between spermatozoa and HL-60 cells suspended in washing medium. In the same way we are able to distinguish between polystyrene beads and spermatozoa. Thus, by adding a known concentration of polystyrene beads to a boar semen sample, the spermatozoa concentrations of seven mixtures are measured and show a good correlation with the actual concentration (R(2)-value = 0.97). To our knowledge this is the first time that the concentration of spermatozoa has been determined on chip using electrical impedance measurements without a need to know the actual flow speed. The proposed method to determine the concentration can be easily applied to other cells. The described on-chip determination of the spermatozoa concentration is a first step towards a microfluidic system for a complete quality analysis of semen.

The micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms.

A miniaturized, disposable microbial culture chip has been fabricated by microengineering a highly porous ceramic sheet with up to one million growth compartments. This versatile culture format, with discrete compartments as small as 7 x 7 mum, allowed the growth of segregated microbial samples at an unprecedented density. The chip has been used for four complementary applications in microbiology. (i) As a fast viable counting system that showed a dynamic range of over 10,000, a low degree of bias, and a high culturing efficiency. (ii) In high-throughput screening, with the recovery of 1 fluorescent microcolony in 10,000. (iii) In screening for an enzyme-based, nondominant phenotype by the targeted recovery of Escherichia coli transformed with the plasmid pUC18, based on expression of the lacZ reporter gene without antibiotic-resistance selection. The ease of rapid, successive changes in the environment of the organisms on the chip, needed for detection of beta-galactosidase activity, highlights an advantageous feature that was also used to screen a metagenomic library for the same activity. (iv) In high-throughput screening of >200,000 isolates from Rhine water based on metabolism of a fluorogenic organophosphate compound, resulting in the recovery of 22 microcolonies with the desired phenotype. These isolates were predicted, on the basis of rRNA sequence, to include six new species. These four applications suggest that the potential for such simple, readily manufactured chips to impact microbial culture is extensive and may facilitate the full automation and multiplexing of microbial culturing, screening, counting, and selection.

Pressure-driven ballistic Kelvin's water dropper for energy harvesting.

In this paper, we introduce a microfluidic-based self-excited energy conversion system inspired by Kelvin's water dropper but driven by inertia instead of gravity. Two micro water jets are produced by forcing water through two micropores by overpressure. The jets break up into microdroplets which are inductively charged by electrostatic gates. The droplets land on metal targets which are gradually charged up to high voltages. Targets and electrostatic gates are cross-connected in a way similar to Kelvin's water dropper. Application of pressure as driving force instead of gravity as in Kelvin's dropper allows for much higher energy densities. To prevent overcharging of the droplets by the inductive mechanism and consequent droplet loss by repulsion from the target as in Kelvin's water dropper, a voltage divider using inversely connected diodes was introduced in our system to control the charge induction providing self-limiting positive feedback by the diode characteristics. A maximal 18% energy conversion efficiency was obtained with the diode-gated system.

High-efficiency ballistic electrostatic generator using microdroplets.

The strong demand for renewable energy promotes research on novel methods and technologies for energy conversion. Microfluidic systems for energy conversion by streaming current are less known to the public, and the relatively low efficiencies previously obtained seemed to limit the further applications of such systems. Here we report a microdroplet-based electrostatic generator operating by an acceleration-deceleration cycle ('ballistic' conversion), and show that this principle enables both high efficiency and compact simple design. Water is accelerated by pumping it through a micropore to form a microjet breaking up into fast-moving charged droplets. Droplet kinetic energy is converted to electrical energy when the charged droplets decelerate in the electrical field that forms between membrane and target. We demonstrate conversion efficiencies of up to 48%, a power density of 160 kW m(-2) and both high- (20 kV) and low- (500 V) voltage operation. Besides offering striking new insights, the device potentially opens up new perspectives for low-cost and robust renewable energy conversion.

A low-cost 2D fluorescence detection system for µm sized beads on-chip.

In this paper we describe a compact fluorescence detection system for on-chip analysis of beads, comprising a low-cost optical HD-DVD pickup. The complete system consists of a fluorescence detection unit, a control unit and a microfluidic chip containing microchannels and optical markers. With these markers the laser beam of the optical pickup can be automatically focused at the centre of the microchannel. With the complete system a two-dimensional fluorescent profile across the channel width can be obtained such that there is no need for hydrodynamic or electrokinetic focusing of the particles in a specific part of the channel. Fluorescent µm sized beads suspended in medium have been detected with the system. Since on both sides of the main beam two additional laser beams at a known distance are generated, also the velocity of individual beads has been determined.

A new floating electrode structure for generating homogeneous electrical fields in microfluidic channels.

In this article a new parallel electrode structure in a microfluidic channel is described that makes use of a floating electrode to get a homogeneous electrical field. Compared to existing parallel electrode structures, the new structure has an easier production process and there is no need for an electrical connection to both sides of the microfluidic chip. With the new chip design, polystyrene beads suspended in background electrolyte have been detected using electrical impedance measurements. The results of electrical impedance changes caused by beads passing the electrodes are compared with results in a similar planar electrode configuration. It is shown that in the new configuration the coefficient of variation of the impedance changes is lower compared to the planar configuration (0.39 versus 0.56) and less dependent on the position of the beads passage in the channel as a result of the homogeneous electrical field. To our knowledge this is the first time that a floating electrode is used for the realization of a parallel electrode structure. The proposed production method for parallel electrodes in microfluidic channels can easily be applied to other applications.