Boris Gorbovitski

A family of novel DNA sequencing instruments based on single-photon detection

We have developed a family of high‐performance capillary DNA sequencing instruments based on a novel multicolor fluorescent detection technology. This technology is based on two technical innovations: the multilaser excitation of fluorescence of labeled DNA fragments and the “color‐blind” single‐photon detection of modulated fluorescence. Our machines employ modern digital and broadband techniques that are essential for achieving superior instrument performance. We discuss the design and testing results for several versions of the automated single lane DNA sequencers, as well as our approach to scaling up to multilane instruments.

Single Photon Counting Module Based on Large Area APD and Novel Logic Circuit for Quench and Reset Pulse Generation

We present the design, implementation, and characterization of a single-photon counting module (SPCM) based on large-area avalanche photodiode (APD) and new logic circuit based on TTL integrated circuits (ICs) for generating precise quench and reset delays. Low dark count rate, high linearity of 2 MHz, maximum dynamic range of 12 MHz, and minimum dead time of 35 ns have been achieved with 0.2 mm peltier-cooled single photon avalanche diode (SPAD) [model C30902S-DTC, Perkin Elmer Optoelectronics (PKI)]. The developed module was fiberized and tested for the detection of fluorescently labeled DNA sequences. Detection sensitivity at the level of single fluorescent molecule has been demonstrated.

Detection of multi-color fluorescent objects with single photon spectrometer

Single photon counting is the most sensitive and accurate method for detection of very weak fluorescent signals obtained in many applications such as DNA sequencing, detection of biological reporters on micro-beads, detection of droplets in micro-fluidic systems, etc. In this paper we describe the use of single photon spectrometer for detection and characterization of very weak multicolor fluorescence produced by mixtures of various fluorescent dyes and quantum dots.

32-channel single photon counting module for ultrasensitive detection of DNA sequences

We continue our work on the design and implementation of multi-channel single photon detection systems for highly sensitive detection of ultra-weak fluorescence signals, for high-performance, multi-lane DNA sequencing instruments. A fiberized, 32-channel single photon detection (SPD) module based on single photon avalanche diode (SPAD), model C30902S-DTC, from Perkin Elmer Optoelectronics (PKI) has been designed and implemented. Unavailability of high performance, large area SPAD arrays and our desire to design high performance photon counting systems drives us to use individual diodes. Slight modifications in our quenching circuit has doubled the linear range of our system from 1MHz to 2MHz, which is the upper limit for these devices and the maximum saturation count rate has increased to 14 MHz. The detector module comprises of a single board computer PC-104 that enables data visualization, recording, processing, and transfer. Very low dark count (300-1000 counts/s), robust, efficient, simple data collection and processing, ease of connectivity to any other application demanding similar requirements and similar performance results to the best commercially available single photon counting module (SPCM from PKI) are some of the features of this system.

Ultra sensitive sensor with enhanced dynamic range for high speed detection of multi-color fluorescence radiation.

This paper describes design of the new ultra sensitive sensor system for fluorescence detection applications. System comprises two units: optical spectra separation unit and detection unit. Optical unit of the sensor performs spatial spectra separation of signal from the laser excited fluorescence, and resulting spectra is collected in the detection part of the system. Optical part is built using diffraction grating as spectra separation element. Detection part comprises 32-channel photomultiplier tube working in single photon counting mode with our 32-channel amplifier. Using single photon detection technique and specific signal processing algorithms for collected data, the proposed system allows to achieve unique combination of characteristics--high sensitivity, high detection speed and wide linearity dynamic range comparing to existing commercial instruments. DNA sequencing experiments with new sensor as detection device, and using two types of lasers (Ar-ion and Nd-YAG) were carried out, yielding sequencing traces which have quality factor of 20 for read lengths as long as 650 base pairs.

Analysis of DNA sequencing systems based on capillary electrophoresis

A method for characterization and optimization of DNA sequencing systems based on capillary electrophoresis is proposed. A model is developed that relates the read length of a sequencing run and the parameters of DNA sequencing systems such as the properties of the separation medium, capillary length, injection time, concentration of the injected DNA sample, detection sensitivity, system noise, and the illumination power. The optimization of DNA sequencing systems will allow determination of optimum regimes for preparation, injection, and separation of DNA samples needed for achieving a read length required. The method proposed is applied to the characterization of capillary-electrophoretic DNA sequencing systems based on single-photon detection.

Highly sensitive single photon detection system for multi-lane DNA sequencer

A multichannel fluorescence detection system for multi-lane DNA sequencer based on single photon counting is presented. The problem of elimination of the lane cross-talk caused by both optical and electronic cross-talk between the detection lanes is discussed. A novel method for elimination of the channel cross-talk is proposed. The calibration procedure for a DNA sequencer with lane cross-talk cancellation is described. Improvement in sensitivity of a DNA sequencer setup by introducing wide collection angle optical system is discussed.

Multi-Color Single Photon Detection With Enhanced Linearity

A new method for multi-color single photon detection with an enhanced linearity is proposed. Due to the use of the multi-channel single photon detection in conjunction with a novel approach to the post processing of the detected signal, the method provides up to an order of magnitude enhancement of the detection linearity and enables an accurate determination of color composition of fluorescence-labeled micro-objects. Application of the new method for the detection of fluorescent microbeads moving in a microfluidic channel is presented.

Enhancing the Linear Dynamic Range in Multi-Channel Single Photon Detector beyond 7OD.

We present design, implementation, and characterization of a single photon detector based on 32-channel photomultiplier tube (PMT) sensor (model H7260-20, Hamamatsu). The developed high-speed electronics enables the photon counting with linear dynamic range (LDR) up to

Single photon spectrometer for high speed detection of multi-color fluorescence radiation

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