G.W.K. van Dedem

Monitoring enzymatic reactions in nanolitre wells.

We have developed a laboratory‐on‐a‐chip microarray system based on nanolitre‐capacity wells etched in silicon. We have devised methods for dispensing reagents as well as samples, for preventing evaporation, for embedding electronics in each well to measure fluid volume per well in real‐time, and for monitoring the fluorescence associated with the production or consumption of NADH in enzyme‐catalysed reactions. Such reactions can be found in the glycolytic pathway of yeast. We describe the design, construction and testing of our laboratory‐on‐a‐chip. We also describe the use of these chips to measure both fluorescence (such as that evidenced in NADH) as well as bioluminescence (such as evidenced in ATP assays). We show that our detection limit for NADH fluorescence is 5 µm with a microscope‐based system and 100 µm for an embedded photodiode system. The photodiode system also provides a detection limit of 2.4 µm for ATP/luciferase bioluminescence.

Fluorescence detection in (sub-)nanoliter microarrays

The goal of our TU Delft interfaculty research program is to develop intelligent molecular diagnostic systems (IMDS) that can analyze liquid samples that contain a variety of biochemical compounds such as those associated with fermentation processes. One specific project within the IMDS program focuses on photon sensors. In order to analyze the liquid samples we use dedicated microarrays. At this stage, these are basically miniaturized micro titre plates. Typical dimensions of a vial are 200 X 200 X 20 micrometer3. These dimensions may be varied and the shape of the vials can be modified with a result that the volume of the vials varies from 0.5 to 1.6 nl. For all experiments, we have used vials with the shape of a truncated pyramid. These vials are fabricated in silicon by a wet etching process. For testing purposes the vials are filled with rhodamine solutions of various concentrations. To avoid evaporation glycerol-water (1:1, v/v) with a viscosity of 8.3 times the viscosity of water is used as solvent. We aim at wide field-of-view imaging at the expense of absolute sensitivity: the field-of-view increases quadratically with decreasing magnification. Small magnification, however, implies low Numerical Aperture (NA). The ability of a microscope objective to collect photons is proportional to the square of the NA. To image the entire microarray we have used an epi-illumination fluorescence microscope equipped with a low magnification (2.5 X/0.075) objective and a scientific CCD camera to integrate the photons emitted from the fluorescing particles in the solutions in the vials. From these experiments we found that for this setup the detection limit is on the order of micromolar concentrations of fluorescing particles. This translates to 108 molecules per vial.

An ISFET-based anion sensor for the potentiometric detection of organic acids in liquid chromatography

An ion-selective field effect transistor (ISFET) was applied as a potentiometric detector in liquid chromatography (LC) for the determination of organic acids. The ISFET was prepared by coating the gate insulator of the encapsulated transistor with a poly(vinyl chloride) (PVC) matrix membrane containing methyltridodecylammoniumchloride, which enables the detection of organic anions. The ISFET was tested for its applicability as detector for carboxylic acids in ion-exchange and reversed-phase chromatography. Its analytical characteristics were compared to those of a coated-wire electrode (CWE) and of a conventional type of ion-selective electrode (ISE).

PCR array on chip - thermal characterization

This paper presents thermal analysis simulation and verification of a 50-nanoliter-reactor PCR (Polymerase Chain Reaction) well for application in silicon arrays, allowing 5/spl times/5 chamber matrix to be fitted on a 1cm/sup 2/ square. Every reactor cell is equipped with an integrated heater, temperature sensor and a photodetector. Each well forms a separate unit independently controlled and thermally insulated from the rest. Through micromachining the thermal capacity of each chamber is minimized, enabling rapid (8 - 10 cycles per minute) PCR cycling. To characterize the thermal behavior, an equivalent lumped element electrical circuit was defined and the results were compared to those obtained by Finite Element Method (FEM) analysis with CoventorWare/spl trade/. The proposed structure was implemented on a silicon substrate using a standard CMOS process and post-processing. Experiments were performed for verification of the model. Analysis shows that a temperature of about 95/spl deg/C can be reached (starting from 55/spl deg/C) by applying 1.5 W of electrical power in the integrated heater over a period of less than 2.5 seconds. The cooling (not active - self cooling) of the device is done in about 1.5 second.

Integrated sensor arrays for bioluminescence and fluorescence bio-chemical analysis

We present on-chip luminescence and fluorescence bio-chemical analysis, using integrated photodiodes. The detectors and the read-out electronics are implemented on a silicon substrate using standard CMOS processing. The photosensitive structures result from two-stacked PN junctions and an (optional) optical filter. The bioluminescent analyses are based on a light producing reaction - the conversion of ATP (adenosine triphosphate) molecules to AMP - catalyzed by the enzyme luciferase. The obtained results for three different initial concentrations of ATP molecules, in ATP consuming reactions, are presented. Initial fluorescent measurements have been conducted, based on the enzyme protein tyrosine phosphatase (PTP1B) using molecular probes DiFMUP (UV excitable). An enzyme solution (500 pg//spl mu/l) was mixed with DiFMUP. The reaction product DiFMU exhibits excitation/emission maxima of /spl sim/358/455 nm. The undesired excitation (UV) light was filtered out with. an integrated on-chip high pass filter with wavelength cut-off at 400 nm.

Monitoring Enzyme-catalyzed Reactions in Micromachined Nanoliter Wells using a Conventional Microscope based Microarray Reader

Yeast-Saccharomyces cerevisiae - it widely used as a model system for other higher eukaryotes, including man. One of the basic fermentation processes in yeast is the glycolytic pathway, which is the conversion of glucose to ethanol and carbon dioxide. This pathway consists of 12 enzyme-catalyzed reactions. With the approach of microarray technology we want to explore the metabolic regulation of this pathway in yeast. This paper will focus on the design of a conventional microscope based microarray reader, which is used to monitor these enzymatic reactions in microarrays. These microarrays are fabricated in silicon and have sizes of 300 by 300 micrometers 2. The depth varies from 20 to 50 micrometers . Enzyme activity levels can be derived by monitoring the production or consumption rate of NAD(P)H, which is excited at 360nm and emits around 450nm. This fluorophore is involved in all 12 reactions of the pathway. The microarray reader is equipped with a back-illuminated CCD camera in order to obtain a high quantum efficiency for the lower wavelengths. The dynamic range of our microarray reader varies form 5(mu) Molar to 1mMolar NAD(P)H. With this microarray reader enzyme activity levels down to 0.01 unit per milliliter can be monitored. The acquisition time per well is 0.1s. The total scan cycle time for a 5 X 5 microarray is less than half a minute. The number of cycles for a proper estimation of the enzyme activity is inversely proportional to the enzyme activity: long measurement times are needed to determine low enzyme activity levels.

Sensorized nanoliter reactor chamber for DNA multiplication

This paper presents thermal analysis verification of a sensorized 50 nl reactor chamber for DNA amplification based on PCR (polymerase chain reaction). The reactor is equipped with an integrated heater, temperature sensor and a photo detector for real time detection. Through micromachining, the thermal capacity of each chamber is minimized, enabling rapid PCR cycling. The proposed structure was implemented on a silicon substrate using a standard CMOS process and postprocessing. The chambers have a bottom area of 500/spl times/500 /spl mu/m/sup 2/ and a pitch of 1 mm. An array of 96 reactors can be formed on a square centimeter. In order to reach the required PCR temperature levels (55/spl deg/C, 75/spl deg/C and 92/spl deg/C) dedicated electronics, based on a proportional-integral (PI) controller were designed and built. The system is capable of stabilizing the temperature of the reactor and performing a temperature sweep up to 100/spl deg/C.

Monitoring enzymatic reactions with in situ sensors

In previous publications and presentations we have described our construction of a laboratory-on-a-chip based on nanoliter capacity wells etched in silicon. We have described methods for dispensing reagents as well as samples, for preventing evaporation, for embedding electronics in each well to measure fluid volume per well in real-time, and for monitoring the production or consumption of NADH in enzyme-catalyzed reactions such as those found in the glycolytic pathway of yeast. In this paper we describe the use of light sensors (photodiodes) in each well to measure both fluorescence (such as that evidenced in NADH) as well as bioluminescence (such as evidenced in ATP assays). We show that our detection limit for NADH fluorescence in 100 μM and for ATP/luciferase bioluminescence is 2.4 μM.

Microinjection of sigma-D-glucose standards and Amplex Red reagent on micro-arrays

Intelligent Molecular Diagnostic Systems (IMDS)- The objective of this multidisciplinary research program is to design and develop an analytical system that is able to measure and interpret concentrations of various analytes which are dispensed on a micro-array. The analytes are detected by means of fluorescence or (chemi)luminescence measurement. Furthermore, the collected data are combined and interpreted using modern reasoning techniques. Micro-injection- Dispensing picoliters (pl) of reagents (enzymes, antibodies, etc.) and liquid samples on a micro-array requires special techniques. At the moment we are working on a technique which will allow for accurately dispensing liquid volumes less than 100 pl on a micro-array. Detection of (beta) -D-glucose- (beta) -D-glucose standards are dispensed on a micro-array, after which a solution of Amplex Red reagent, horse radish peroxidase (HARP), and glucose oxidase in a mixture of ethylene glycol and water is added. Ethylene glycol is added to prevent evaporation. The (beta) -D-glucose reacts with glucose oxidase to D-gluconolactone and H2O2. The H2O2 reacts with 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) with a 1:1 stoichiometry to produce highly fluorescent resorufin. The formation of resorufin with time is followed with a Zeiss Axioskop microscope equipped with a KAF Photometrics CCD camera, in order to determine the sensitivity, concentrations, and volumes associated with the dispensed fluids.

Spatially Localized Voltage Control in Glass Microchannels for Enhanced Sample Handling Flexibility

Electro-osmotically induced pumping (EOIP) can be used for sample and fluid handling in field-free regions in microfluidic devices. Here, EOIP for fluid handling in field-free regions is combined with electrophoretic separation, resulting in enhanced flexibility in sample handling. The influence of the EOIP-driven sample zone formation on the resulting peak area and separation efficiency was investigated. Increased EOIP voltages resulted in the injection of larger sample zones, as indicated by increased peak areas and decreased separation efficiency.