Ventzeslav P. Iordanov

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.

Customized CMOS wavefront sensor

We report on an integrated Hartmann wavefront sensor (WFS) using passive-pixel architecture and pixels clustered as position-sensitive detectors for dynamic wavefront analysis. This approach substitutes a conventional imager, such as a CCD or CMOS imager, by a customized detector, thus improving the overall speed performance. CMOS (complementary-metal- oxide-semiconductor) technology enables on-chip integration of several analog and digital circuitry. The sensor performance depends on the feature size of the technology, noise levels, photosensitive elements employed, architecture chosen and reconstruction algorithm.

Filter-protected photodiodes for high-throughput enzymatic analysis

This paper relates to the use of a thin film of re-crystallized (polycrystalline) silicon as a low-pass rejection filter in the ultraviolet light range and, more particularly, to the use of this layer as a protective layer for semiconductor diodes. The polycrystalline silicon filters were fabricated by laser annealing a thin film of amorphous silicon deposited by an LPCVD process. A standard component of the polysilicon-gate CMOS process is the boron phosphor silicate glass (BPSG) planarization layer. Since this layer is always applied, the possibility of using it as the isolator between the diode and the filter (and, thereby, omit one SiO/sub 2/ layer) is considered. Using scanning electron microscopy, we compared the crystallization process of the LPCVD silicon film deposited on a glass substrate and on a BPSG layer. The fabrication and the characterization of the filter-protected photodiodes are described in the paper.

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.

Some properties of a room temperature THz detection array

Detection peculiarities of an un-cooled (room temperature) 8x8 pixel array designed to image broadband THz radiation were investigated. Each pixel consists of a thin conductive film absorber on a dielectric membrane with thermopile temperature readout. It was designed and tested for four combinations of two different types of absorber and thermopile materials. The photo-response profile, determined by scanning the pixels through the focus of a THz laser beam, was wider than expected from a 2-D convolution of the Gaussian beam and the absorber surface. Also the time response did depend on the position of the beam relative to the pixel. Simulations show that those properties are due to the fact that also the thermopiles absorb THz radiation. For the best composition of absorber and thermopile, the responsivity, the noise equivalent power, and the bandwidth were estimated to be of 28 V/W, 5x10-9 W/Hz1/2 and 50 Hz, respectively.

CMOS-compatible wells for integrated high-speed screening arrays

This paper explores the use of photo patternable polymers for integrated high-speed screening arrays, where enzyme reactions are monitored in nano liter volume reactors using fluorescence of NADH and photodiode detection. Implementing the array of nano liter volume wells using a low-temperature CMOS-compatible process allows wells to be patterned after the photodiode array and electronics fabrication is completed. We demonstrate filling of 400 X 400 micron square, 25 micron deep photoresist-on-silicon wells with liquid samples by electro spray and wetting. We also demonstrate usability of the wells on NADH samples by measuring the fluorescence of 0.1, 0.5 and 1 millimolar NADH solutions using external optics.

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.

Nanoliter array advances: miniaturized, high-speed PCR sensing & control

We have previously reported on our laboratory-on-a-chip nanoarray system based on nanoliter-capacity wells etched in silicon (Young, I.T. et al., J. Microscopy, vol.212, no.3, p.254-63, 2003; Dietrich, H.R.C. et al., Analytical Chemistry, vol.76, no.14, p.4112-17, 2004). We now describe how temperature sensing and control embedded in the floor of the nano-wells make it possible for us to cycle each well independently through the temperatures 92/spl deg/C, 55/spl deg/C, and 75/spl deg/C. This individual temperature cycling on nanoliter wells means that the nano-array architecture is suitable for PCR applications and that the total time needed for 30 cycles of PCR amplification could be less than five minutes. Further, we describe how, by embedding photodiodes in the floor of the wells, we can track the fluorescence associated with the melting and annealing of DNA when labeled with a suitable nucleic acid stain. Measurements performed with the fluorophores Rhodamine B and SYBR Green I have demonstrated our ability to control the temperature, measure the fluorescence, and monitor the denaturation and renaturation of DNA.

Si Based Thin-Film Filter with High Visible-Over-UV Selectivity for Biochemical Fluorescence Analysis

In this paper we describe a new optical detector system for fluorescence analysis in on-chip high-speed-screening arrays. It consists of a photodiode covered by a re-crystallized silicon film, which acts as a single-film optical filter. The fabrication is fully compatible with standard CMOS processes. The optical properties of the filter are such that fluorescent light of wavelengths of 420 nm or higher can be measured in the presence of the (UV) excitation light (340–360 nm). A selectivity exceeding 35 dB has been demonstrated. The technique has the potential for 50 dB selectivity and higher. NADH concentration measurements illustrate the application of the system for determination of enzymatic activity of different analytes.