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Portable genotyping system: Four-colour microchip electrophoresis
Sensors and Actuators B 143 (2010) 583–589
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Portable genotyping system: Four-colour microchip electrophoresis Ivan Rech ∗ , Stefano Marangoni, Angelo Gulinatti, Massimo Ghioni, Sergio Cova Politecnico di Milano, Dipartimento di Elettronica e Informazione, Piazza L. da Vinci 32 - 20133 Milano, Italy
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Article history: Received 12 February 2009 Received in revised form 28 September 2009 Accepted 30 September 2009 Available online 9 October 2009 Keywords: Genotyping DNA sequencing Single photon avalanche diode SPAD array Photon counting
a b s t r a c t In the field of genomic research, the microcapillary electrophoresis (MCE) plays a key role, allowing a small reagent use but requiring the ability to detect luminescences down to a few photons per second. Very high sensitivity detectors are therefore required, rising the systems’ final costs and reducing their diffusion. There is a strong request to develop compact and low cost systems. The state of the art for MCE-based DNA analysis instruments is the use of photomultiplier tubes (PMTs) or CCDs, but either have some drawbacks. As for the PMTs, they cannot be integrated, so detection of the four bases can occur only using four different PMTs, increasing size and cost. As for the CCDs, even if they are commonly used, they are not the best solution regarding simplicity and cost. A valid alternative is single photon avalanche diodes (SPADs). Such devices present high quantum efficiency (higher than PMTs), with the possibility to integrate several detectors on a single chip, even with custom geometries, retaining low fabrication costs and high sensitivity. The strong potential of planar SPAD technology for DNA analysis has already been demonstrated in previous works. Here we present the first genotyping system based on a custom array of four SPADs, purposely designed to be used in conjunction with Beckman-Coulter markers. The module size is 19 cm (L), 12.5 cm (W), 15 cm (H), including high voltage section, detectors and optical system: at the best of our knowledge, this is the most compact four-colour genotyping system in literature. © 2009 Elsevier B.V. All rights reserved.
1. Introduction A great number of decisions in the modern society are based on genetic analysis data and in the next years the demand of this type of information will grow in exponential way pushing the technology toward new goals in terms of performances. In the field of DNA analysis, sequencing techniques play a fundamental role in genotyping. Through this approach it is possible to determine an individual’s full genotype, that is the collection of genes determining a particular phenotypic trait. Study of these traits is very important, since several diseases are caused by alteration of a single nucleotide in a specific position in the genome, so through genotyping a better understanding of these diseases can be obtained. The most common genotyping technique is laser-induced fluorescence (LIF) with microcapillary electrophoresis (MCE), that guarantee high sensitivity [1,2] but often requires bulky and costly apparatus. Compact low-cost analysis systems are instead required to perform simple, fast and low-cost tests wherever necessary, even in small laboratories [3]. The miniaturization of the separation process has a number of advantages, which include low reagent consumption, integration of a series of analytical steps, rapid analysis and relative low unit cost but require to detect and measure
∗ Corresponding author. Tel.: +39 02 2399 3700; fax: +39 02 2367 604. E-mail address: [email protected] (I. Rech). 0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2009.09.058
the fluorescence from smaller and smaller samples, with ultraweak intensity, down to a few photons per second. High sensitivity detection systems in ultracompact apparatus are thus required. The state of the art [4–6] for MCE-based DNA analysis instruments makes use of photomultiplier tubes (PMTs) or highsensitivity CCDs. Very high PMT internal gain (105 ) produces high output pulses and makes possible to detect even single photons; their wide sensitive area (diameters from 10 mm to various centimeters) simplifies the design of the optical systems. However, PMTs present some drawbacks that prevent their use in compact genotyping systems: they cannot be integrated, so detection of the four bases can occur only using four different PMTs, increasing size and cost. Finally, PMTs have lower quantum efficiency compared to solid-state detectors, especially in the red and near-infrared spectrum. In recent years high-sensitivity CCDs have been employed [7]. They present very high quantum efficiency and, cooled to very low temperatures, very low noise as well [8]. Such low temperatures (about −50 ◦ C) are obtained using air or a liquid coolant, resulting in a bigger and more delicate apparatus. In summary, even if CCDs are commonly used in DNA sequencing, they are not the best solution regarding simplicity, cost and dimensions. The ideal detection head for low cost genotyping systems for large scale distribution should consist of only 4 pixels, one for each wavelength, capable of detecting single photons to properly detect even short phenomena, lasting for a few tens of milliseconds. For CCDs this is generally
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voltage. The bias voltage is then restored in order to detect another photon. This operation requires a suitable circuit that is usually referred to as quenching circuit [10,11]. SPADs have good quantum efficiency, better than PMTs, and all the advantages of solid-state devices (miniaturization, ruggedness, low voltage, low power, and reduced cost). Therefore a monolithic array of SPAD detectors could represent a breakthrough for the evolution of genotyping systems, because it allows to combine very high sensitivity with extreme compactness and simple system architecture, thanks to the low number of pixels to manage and the moderate cooling (−15 ◦ C) necessary for the best performances. The whole optical detection head, including the array detector and the associated electronics are described in Section 2. The structure and the fabrication process of the array detector are described in Section 2.3, while in Section 3 the system performances are reported. 2. Materials and methods
Fig. 1. LIF-MCE apparatus for DNA analysis; (a) scheme of the proposed apparatus, (b) photograph of the apparatus.
LIF-MCE genotyping systems are based on chain termination method [12] developed by Frederick Sanger in 1977. This method first involves breaking chromosomes, which range in size from 50 million to 250 million bases, into much shorter pieces. Each of this is used as a template to generate a set of fragments that differ in length by a single base, that will be identified in a later step, thanks to the labeling of each dideoxynucleotide chain-terminators with a different fluorescent dye. The fragments in a set are separated by microchip electrophoresis, i.e. the movement of an electrically charged substance under the influence of an electric field. Due to their size, fragments have different frictional forces, so they will separate from one another as they migrate through a buffer solution. Identifying the final base at the end of each fragment, it’s possible to recreate the original sequence of As, Ts, Cs, and Gs for each short piece generated in the first step. Thus, a system for DNA sequencing or genotyping should include (1) an ultra-stable high voltage section to supply correct voltages for the electrophoretic separation, (2) one or more laser to excite fluorescence of the different markers, (3) an optical system that focuses laser light on separation channel, collects fluorescence signals and allows the distinction of the four different fluorescence wavelengths, and (4) a detection unit composed at least of four pixels, one for each marker, with very high sensitivity, down to few photons per second. Moreover, a movement unit is required to automatically align the capillary with the laser spot. In Fig. 1(a) is shown a scheme of the apparatus here proposed. The LIF module size is 19 cm (L), 12.5 cm (W), 15 cm (H) (Fig. 1(b)). 2.1. Fluorescence excitation and electrophoretic separation
difficult, since in short integration times the noise relative to single pixels is no longer due to thermal noise but to reading noise independent of integration time. In this paper we present a complete system based on a custom solid-state single photon avalanche diode (SPAD) array detector. These microelectronic devices join the advantage of PMT and CCD solution. A SPAD is essentially a p-n junction operated in Geigermode, i.e. biased at a voltage higher than the breakdown [9]. In this operating regime, a single carrier generated in the depletion region can trigger a self-sustaining avalanche current. The current swiftly rises with nanosecond or subnanosecond risetime to a macroscopic steady level in the milliampere range, which can be easily discriminated. If the primary carrier is photogenerated, the leading edge of the avalanche pulse marks the arrival time of the detected photon. After the avalanche is triggered, the current keeps flowing until the avalanche is quenched by lowering the bias below the breakdown
Markers considered for the project of the entire system are Beckman-Coulter biomarkers, that fluoresce at 670, 705, 775 and 810 nm (Fig. 2). The choice of these markers allows the use of two simple, very low-cost and compact laser diode (650 nm and 755 nm) to excite the fluorescence, instead of bulky and costly lasers needed for other types of markers (typically Argon lasers). As for the microchip for the electrophoretic separation of the fragments, a Micronit microchip is used, characterized by very small dimensions (45 mm × 15 mm × 1.8 mm) and a channel width of 50 m. 2.2. Optical system The key elements to achieve high sensitivity are optical system and detection unit. Optical system has to collect emitted light
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focusing fluorescence signal on the detector. Moreover, a dispersing section has to spatially separate fluorescence wavelengths. A detailed diagram of the optical module is shown in Fig. 3(a). The excitation beams from two laser diodes (650 nm and 755 nm) are coupled into a microscope objective with a high numerical aperture, which focuses the beams into a 50 m-spot within the sample. The emerging fluorescence is collected by the same objective and separated from the excitation beams by dichroic mirrors. For a better filtering of the source signals, a series of optical filters are placed after dichroic mirrors. The four remaining wavelengths are spatially separated by a transmission grating (600 l/mm Thorlabs) and then focalized on array detector with a collimating triplet (GLC002 by Melles Griot). The optical filters were provided by Chroma Technology USA. We used a laser diode TOLD 9442 manufactured by Toshiba, which provides up to 5 mW of optical power at 650 nm and a laser diode L7910 manufactured by Hamamatsu capable of an output power of 4 mW at 750 nm. Fig. 2. Markers’ emission spectrum.
2.3. Detection unit: single photon array detector As for the detection unit, it has to be able to detect four fluorescence signals with ultrahigh sensitivity and with short acquisition
Fig. 3. Optical system; (a) block diagram, (b) photograph of the custom optical breadboard.
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Fig. 6. Photon detection efficiency of the SPAD array: due to the good uniformity of the technology process PDE spread is limited to a few percent and the curves overlap.
Fig. 4. Optical simulation used for custom design of array detector; (a) optical simulation, (b) layout of custom design array detector.
time (about 100 ms), in order to give an electropherogram with a good temporal resolution. Since high-performance CCDs have best sensitivity only for long acquisition times and since detector has to collect light from only four spot simultaneously, a simpler and more efficient approach than image acquisition may be sought. Performing simulations of the entire optical system, it was possible to determine (Fig. 4(a)) the exact positions on image plane of the four wavelengths, spatially separated by the transmission grating. Moreover, simulation showed how a certain bandwidth in fluorescence emission spectrum translates in line-width on image plane, as a function of the central wavelength. Here we present a custom designed SPAD array with pitch between pixels and active area diameter specifically designed to efficiently detect Beckman markers’ fluorescence signal (Fig. 4(b)). The choice of active area dimension allows a spatial filtering of non-fluorescence wavelengths. In this work, array detector was designed so that each pixel detects only 10 nm-bandwidth around central fluorescence wavelength in order to reduce the crosstalk between different markers. Fig. 5 shows the cross-section of an individual SPAD element in the array. The double-epitaxy structure provides a low series
Fig. 5. Cross-section of a double-epitaxy SPAD.
resistance for the avalanche current. The p+ boron implantation defines the high field region, where avalanche multiplication takes place. Deep phosphorus diffusions provide full electrical isolation among SPAD elements [13]. A monolithic array of 4 SPAD detectors (Fig. 4(b)) was fabricated on 4 in. wafers at IMM-CNR (Bologna) by using the fabrication process described in ref. [13]. Suitable gettering processes were used to avoid the steep increase of the dark counting rate with the detector diameter that was observed in the previous generations of SPAD devices [14]. Each SPAD has an active area of 50 m × 70 m and the pitch between SPADs is designed to optimize fluorescence collection when Beckman Coulter biomarkers are used (Fig. 2): distances between adjacent couples of devices is 165, 327 and 191 m, respectively. Position of each SPAD determines central detection wavelength, while longitudinal width (70 m) corresponds to a wavelength bandwidth of 10 nm in the worst case. To assess the suitability of the available SPAD arrays to the production of a parallel detection system, fabricated devices were fully characterized. We first tested individually the detectors in the array in order to evaluate their performances; the crosstalk between different detectors was then measured to verify the characteristics of our devices when operated simultaneously. 2.3.1. Photon detection efficiency The photon detection efficiency (PDE) of the array was evaluated operating the SPADs at 5 V excess bias voltage. Its behavior as a function of wavelength is shown in Fig. 6. The efficiency has a peak of about 50% at 550 nm and is about 12% at 820 nm. These values are consistent with the structure of the photon absorption region, which is about 5 m thick. Different pixels in the array show the same shape of the efficiency function, with a spread limited to a few percent. These variations are due both to the spread of the detector overvoltage and to the process non-uniformities over the chip. 2.3.2. Dark counting rate Even without illumination, thermally generated electron–hole pairs can trigger an avalanche in the device, thus producing dark counts. The dark counting rate (DCR) is affected by a Poissonian fluctuation that represents the noise of the detector. Fig. 7 shows the dark counting rates, sorted in increasing order, of the four SPADs in a typical array. Fig. 8 shows the temperature behavior of the DCR for a single SPAD. As a general rule, dark counts are caused by the combined effect of band-to-band tunneling and Shockley Reed Hall generation; this two contributions show a different temperature behavior, with tunneling effect dominating at low temperatures and SRH generation at high temperatures. The electric field in the
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Fig. 7. Dark counting rate.
depletion region of the detector can be engineered to reduce the contribution of band-to-band tunneling, which limits the possibility of reducing the DCR by cooling the device. The curves in Fig. 8 are relative to two different design of the detector. Older devices (“standard” electric field) showed a pronounced tunnel effect that limited the working temperature to −5 ◦ C, because further reduction of the temperature did not reduce significantly the dark counts. The detectors used in our apparatus (“engineered” electric field) were specifically designed to cut the contribution of tunneling, so that the temperature dependence of the DCR is now much steeper [14,15]; its worth noting that, in respect to the older design, the dark count rate is lower even at room temperature. 2.3.3. Crosstalk Crosstalk represents the correlation between the output signals of different channels. Ideally the counting rates of two different detectors in the array should be totally uncorrelated. Unfortunately optical coupling between nearby devices causes some sort of correlation; at the device level this is due to the avalanches that are triggered in one detector as a result of the optical emission of another SPAD that is subject to current flow. Crosstalk effects were evaluated using the time correlated single photon counting (TCSPC) technique; measurements were performed at −10 ◦ C, operating the SPADs at 5 V excess bias voltage and with an holdoff time of 100 ns. Measures show that the crosstalk varies from 8.1 × 10−4 to 1.7 × 10−3 , depending on the distance between the considered detectors. The measured crosstalk does not follow a
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monotonic behavior: it first increases and then smoothly decreases in an unpredicted fashion. This behaviour can be explained considering indirect optical paths reecting off the bottom silicon–air interface where the classical total internal reection takes place for rays above the critical angle. All the photons emitted by a SPAD and reecting at the bottom of the chip with an angle of incidence around (or greater than) the critical angle can reach another device causing optical crosstalk. Conversely, only a fraction of the photons reecting with an angle of incidence smaller than the critical angle can reach another device; for this reason their contribution to the crosstalk is less significant. Since larger distances between SPADs imply greater angles of incidence for the reected paths going from one device to the other, the observed “bell shape” can be ascribed to the overcoming of the critical angle. In our labs it was developed an accurate 3D optical model that takes into account substrate doping level, silicon absorption lengths as a function of wavelength and isolation doping level, considering also secondary effects due to rejection on lateral surfaces [16]. 2.4. Optical alignment In currently used MCE setups, the operator is often requested to perform several manual operations every time the chip is removed, such as washing, refilling, etc. These operations include the alignment of the microchannel with the optical path of the LIF module. In order to avoid a time-consuming manual alignment, we have developed a fully automated procedure. This procedure is based on the measurement of the faint signal generated by a wide-spectrum led light that is scattered from the glass-microchannel discontinuity and is not completely cut-off by the optical filter system. By using a micrometric translation stage, the laser beam is scanned onto the electrophoresis chip over a line perpendicular to the microchannel. An onboard microcontroller moves step-by-step to the position of the translation stage, counts the photon pulses from a SPAD detector at each position, and sends the recorded counts to the PC through a USB interface. The scattered light signal attains its maximum when the microchannel is perfectly aligned to the optical path. The remote PC performs all the computations needed to find out this maximum; then it communicates the result to the microcontroller, which finally aligns the system to the position of the maximum. It is worth stressing that it is the precise alignment guaranteed by the automated system that makes possible to perform the experiments with high repeatability and practically zero failure rate. In fact, since the laser spot diameter and the microchannel section have the same size (approximately 50 m), even a small misalignment (few microns) between them causes a significant reduction of the instrument sensitivity. Due to the large depth of field of the optics, the position along the z-axis is simply adjusted via a vertical translator before carrying out a series of measurements. 2.5. Electronic section and software interface
Fig. 8. Noise temperature behavior of the noise of two different generations of SPAD devices.
To implement the pinched mode injection-separation sequence, three independent high-voltage power supplies are developed using ultra-compact DC-HV DC converter from EMCO. A closed loop system was designed to obtain very stable and precise voltage levels. The maximum output voltage is 4 kV. The currents are monitored during the DNA injection and separation; the available measurement ranges are 0–25, 0–50, and 0–100 A. Platinum electrodes are connected to the microchip reservoirs by means of a servo-mechanical actuator. Chip holder, plexiglass electrode seat and connection system are designed to obtain a very compact system. Each parameter in the apparatus is fully controlled by a PC. The adjustable parameters include the working conditions of the SPAD
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structure allowed a very simple testing of all the parts, but implies a sizable encumbrance of the optical system. To overcome this problem, a different structure has been realized. The apparatus here described is based on a transmission grating as separation element and two optical groups to collect fluorescence light and to focus it on the SPAD array. Assuming that the fluorescence from the microcapillary acts like a Lambertian source it can be note that the power radiation in terms of W/m2 ·sterad is: B(˛) = B(0) · cos(˛) So, the power emitted from the source on a solid angle d is: d = AS · B(˛) · d˝ = AS · B(0) · cos(˛) · d˝ where d and d␣ are related by d˝ = 2 · sin(˛) · d˛ The collection efficiency of the optical system is given by the ratio between incident light with an angle lower than acceptance angle and the total light emitted:
˛max
=
0
/2 0
Fig. 9. Optical dispersion of the realized apparatus.
detectors (bias voltage, dead-time, temperature), and the output power of the excitation laser. It is also possible to perform a series of runs while varying step-by-step a particular parameter, e.g., the laser power or the injection voltage. The instrument is connected to a nearby PC through a USB interface. 3. Results and discussion Optical system and detection unit are the key blocks in the apparatus to achieve high sensitivity. As for the optical system, one of the most important parameters is the true optical dispersion, because this characteristic determines the ability to distinguish and spatially filter different wavelengths. Fig. 9 shows the good agreement between experimental data and optical simulation. To obtain the experimental data, we collected the light coming from transmission grating with a focusing lens GLC002 (Melles Griot). Image on focal plane was acquired with a CCD camera. Spots corresponding to wavelengths with 10 nm bandwidth were realized using an Oriel monochromator. The huge potential of SPAD detector in LIF-MCE DNA analysis was demonstrated in a previous work of our group [17] in which the use of two SPADs as detection head allowed a sensitivity limit of 3 pM with 50 pl injection volume. The apparatus here presented represents the natural evolution of the previous one in terms of compactness and type of analysis, without decreasing the overall performances. In fact, since the optical collection efficiency of the two systems is the same, the apparatus presented is able to perform DNA sequencing and genotyping analysis, with analogous detection limit in extremely compact dimensions. The apparatus described in [17] employed interferential filters to separate fluorescence wavelengths and optical fibers to couple fluorescence light on the corresponding SPAD detector. For that system its possible to derive a peak collection efficiency of 1.6%, considering fluorescence emission from the microcapillary as a lambertian source and accounting for filter’s transmittivity, light fiber coupling and numerical aperture of the collection lens. This
2 · sin(˛) · AS · B(0) · cos(˛) · d˛ 2 · sin(˛) · AS · B(0) · cos(˛) · d˛
=
NA 2 n
where NA = n·sin(˛) is the numerical aperture and n is the refractive index of the material. So the overall optical efficiency, considering the transmission grating transmittivity is given by: = · = 0.16 · 0.35 = 5.6% This value is confirmed by experimental results obtained using an optical fiber as source. This is a good choice to emulate a capillary for LIF-MCE: 50 micron core dimension fit with typical cross-section of microchip capillary and its possible to generate test wavelength with a monochromator in all visible range, covering most of possible marker’s fluorescence wavelengths. In the next months we will perform a full set of DNA analysis to validate our apparatus, but since the system here presented and the apparatus described in [17] have comparable optical efficiency, we are confident that even the detection limit will be comparable. 4. Concluding remarks The development of an ultra-compact instrument based on single photon avalanche diode (SPAD) and able to perform MCE DNA sequencing analysis is here reported. The planar epitaxial SPAD combine the typical advantages of microelectronic devices (miniaturization, ruggedness, low voltage, low power, low cost, etc.) with high sensitivity. The suitability of such SPAD to MCE has been demonstrated in recent works. The apparatus presented here could represent a breakthrough in the development of genetic diagnostic system thanks to the extreme compactness and the low cost of basic components. At the best of our knowledge, it is the most compact system for genotyping reported in literature and it could open the way to the development of a new category of genotyping systems, portable and low-cost. The intrinsic performance of the system was tested demonstrating a good fitting with simulations. A comparison with other assays will be accomplished in the next months. Since this system is the natural evolution of a former work (supported by the Italian Ministry of University and Research within the framework of the Project FIRB No. RBNE01SLRJ\ 001 “Microsystems for genetic diagnostics”) that brought to a DNA separation system with sensitivity better
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than 3 pM, we are confident that the system will show a sensitivity comparable with the best DNA sequencers. References [1] E. Verpoorte, Microfluidic chips for clinical and forensic analysis, Electrophoresis 23 (2002) 677–712. [2] N.J. Dovichi, J. Zhang, K.O. Voss, D.F. Shaw, et al., A multiple-capillary electrophoresis system for small-scale DNA sequencing and analysis, Nucleic Acids Research 27 (1999) 24. [3] T. Vo-Dinh, B. Cullum, Biosensors and biochips: advances in biological and medical diagnostics, Fresenius Journal of Analytical Chemistry 366 (6–7) (2000) 540–551. [4] Q. Gao, Y. Shi, S. Liu, Multiple-channel microchips for high-throughput DNA analysis by capillary electrophoresis, Fresenius Journal of Analytical Chemistry 371 (2001) 137–145. [5] L. Alaverdian, S. Alaverdian, O. Bilenko, I. Bogdanov, E. Filippova, D. Gavrilov, B. Gorbovitski, M. Gouzman, G. Gudkov, S. Domratchev, O. Kosobokova, N. Lifshitz, S. Luryi, V. Ruskovoloshin, A. Stepoukhovitch, M. Tcherevishnick, G. Tyshko, V. Gorfinkel, A family of novel DNA sequencing instruments based on singlephoton detection, Electrophoresis 23 (16) (2002) 2804–2817. [6] A. Stepukhovich, A. Tsupryk, O. Kosobokova, D.N. Gavrilov, B. Gorbovitski, G. Gudkov, G. Tyshko, M. Tcherevishnik, V. Gorfinkel, Analysis of DNA sequencing systems based on capillary electrophoresis, Technical Physics 53 (6) (2008) 763–775. [7] D. Meldrum, Automation for genomics, part two: sequencers, microarrays, and future trends, Genome Research 10 (2000) 1288–1303. [8] J.R. Janesick, Scientific Charge-Coupled Devices, SPIE Press Monograph, 2001. [9] M. Ghioni, A. Gulinatti, I. Rech, F. Zappa, et al., Progress in silicon single-photon avalanche diodes, IEEE Journal of Selected Topics in Quantum Electronics 13 (4) (2007) 852–862. [10] S. Cova, M. Ghioni, A. Lacaita, C. Samori, et al., Avalanche photodiodes and quenching circuits for single photon detection, Applied Optics 35 (1996) 1956. [11] A. Gallivanoni, I. Rech, D. Resnati, M. Ghioni, et al., Monolithic active quenching and picosecond timing circuit suitable for large–area single–photon avalanche diodes, Optics Express 14 (12) (2006) 5021–5030. [12] F. Sanger, S. Nicklen, A.R. Coulson, DNA sequencing with chain-terminating inhibitors, Proceedings of the National Academy of Sciences of the United States of America 74 (12) (1977) 5463–5467. [13] A. Lacaita, M. Ghioni, S. Cova, Double epitaxy improves single-photon avalanche diode performance, Electron. Lett. 25 (1989) 841–843. [14] M. Ghioni, A. Gulinatti, P. Maccagnani, I. Rech, et al., Planar silicon spads with 200 (m diameter and 35 ps photon timing, in: Proceedings of SPIE, Optics East 2006, Advanced Photon Counting Techniques 6372, Boston, MA, USA, 2006. [15] M. Ghioni, A. Gulinatti, I. Rech, P. Maccagnani, et al., Large-area low-jitter silicon single photon avalanche diodes, paper presented at SPIE Photonics West 2008—Quantum Sensing and Nanophotonic Devices V, San Jose, CA, USA, January 19–24, 2008. [16] I. Rech, A. Ingargiola, R. Spinelli, I. Labanca, et al., Optical crosstalk in single photon avalanche diode arrays: a new complete model, Optic Express 16 (12) (2008) 8381–8394. [17] I. Rech, S. Cova, A. Restelli, M. Ghioni et al., Microchips and single-photon avalanche diodes for DNA separation with high sensitivity, Electrophoresis 27 (19) (2006).
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Biographies Ivan Rech, born in Milan in 1976, graduated in 2000 in Electronic Engineer at Politecnico di Milano, where he is currently Assistant Professor. PhD in Information Technology at Politecnico di Milano in 2004. His current research interests are focused on the development of single photon detectors and associated electronics for new microanalytical techniques in biomedical, genetic and diagnostic applications. He developed a photon detection module with high timing resolution, which has been employed in florescence measurements on single molecules at the Department of Chemical Biology, Harvard University. He was involved in several interdisciplinary research projects on molecular biology, quantum cryptography, adaptive optics in astronomy, developing dedicated instrumentation and electronic devices. Stefano Marangoni is Ph.D. Student in Information Technology at the Politecnico di Milano, Italy. He has been working on the development of ultra-compact electronic systems for very faint luminescence analysis with single photon photodetectors. His work is focused on complete SPAD array-based systems, ranging from power electronic for CE separation processes and digital programmable electronic development to conjugated optical system project and in-depth analysis of optical crosstalk in detector arrays. Angelo Gulinatti was born in Codogno, Italy, in 1977. He received the Laurea degree in electronic engineering and the Ph.D. degree in information technology (both summa cum laude) from Politecnico di Milano, Milan, Italy in 2003 and 2007, respectively. Since 2008 he is Assistant Professor at Politecnico di Milano where he carries out his research activity in the development and modeling of silicon single photon avalanche diodes (SPAD). Massimo Ghioni (M’91) received the Laurea degree (cum laude) in Nuclear Engineering from the Politecnico di Milano, Italy in 1987. Since 1990, he has been with the Department of Electronics and Information of the Politecnico di Milano, where he is currently Full Professor of Electronics. In 1992 he was visiting scientist at the IBM T.J. Watson Research Center, Yorktown Heights, NY, USA, where he developed a new CMOS compatible SOI photodetector for optical datacom applications. His current research interests are focused on the development of SPAD detectors and associated electronics for applications in physics, astronomy, and molecular biology. He is author of over eighty papers in international refereed journals and conferences and of six U.S. and European patents. Dr. Ghioni received the AEI (Italian Association of Electrical and Electronic Engineers) Award in 1996 and the IEEE PELS Transactions Prize Paper Award for 2004. Sergio Cova, born in 1938 Roma, Italy, he received a doctor degree in Nuclear Engineering in 1962 from Politecnico di Milano, Italy, where he is Full professor of Electronics since 1976. Fellow of the IEEE, he is author of over a 170 papers in international refereed journals and conferences and of five international patents (USA and Europe). He has given innovative contributions in the research and development of detectors for optical and ionising radiations and associated electronics, of microelectronic devices and circuits, of electronic and optoelectronic measurement instrumentation and systems. He pioneered the development of single photon avalanche diodes SPAD, inventing the active-quenching circuit AQC, which opened the way to their application, and devising new SPAD device structures. He collaborated to interdisciplinary research in physics, astronomy, cytology and molecular biology, developing dedicated electronic and optoelectronic devices and instrumentation.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Portable genotyping system: Four-colour microchip electrophoresis Ivan Rech ∗ , Stefano Marangoni, Angelo Gulinatti, Massimo Ghioni, Sergio Cova Politecnico di Milano, Dipartimento di Elettronica e Informazione, Piazza L. da Vinci 32 - 20133 Milano, Italy
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Article history: Received 12 February 2009 Received in revised form 28 September 2009 Accepted 30 September 2009 Available online 9 October 2009 Keywords: Genotyping DNA sequencing Single photon avalanche diode SPAD array Photon counting
a b s t r a c t In the field of genomic research, the microcapillary electrophoresis (MCE) plays a key role, allowing a small reagent use but requiring the ability to detect luminescences down to a few photons per second. Very high sensitivity detectors are therefore required, rising the systems’ final costs and reducing their diffusion. There is a strong request to develop compact and low cost systems. The state of the art for MCE-based DNA analysis instruments is the use of photomultiplier tubes (PMTs) or CCDs, but either have some drawbacks. As for the PMTs, they cannot be integrated, so detection of the four bases can occur only using four different PMTs, increasing size and cost. As for the CCDs, even if they are commonly used, they are not the best solution regarding simplicity and cost. A valid alternative is single photon avalanche diodes (SPADs). Such devices present high quantum efficiency (higher than PMTs), with the possibility to integrate several detectors on a single chip, even with custom geometries, retaining low fabrication costs and high sensitivity. The strong potential of planar SPAD technology for DNA analysis has already been demonstrated in previous works. Here we present the first genotyping system based on a custom array of four SPADs, purposely designed to be used in conjunction with Beckman-Coulter markers. The module size is 19 cm (L), 12.5 cm (W), 15 cm (H), including high voltage section, detectors and optical system: at the best of our knowledge, this is the most compact four-colour genotyping system in literature. © 2009 Elsevier B.V. All rights reserved.
1. Introduction A great number of decisions in the modern society are based on genetic analysis data and in the next years the demand of this type of information will grow in exponential way pushing the technology toward new goals in terms of performances. In the field of DNA analysis, sequencing techniques play a fundamental role in genotyping. Through this approach it is possible to determine an individual’s full genotype, that is the collection of genes determining a particular phenotypic trait. Study of these traits is very important, since several diseases are caused by alteration of a single nucleotide in a specific position in the genome, so through genotyping a better understanding of these diseases can be obtained. The most common genotyping technique is laser-induced fluorescence (LIF) with microcapillary electrophoresis (MCE), that guarantee high sensitivity [1,2] but often requires bulky and costly apparatus. Compact low-cost analysis systems are instead required to perform simple, fast and low-cost tests wherever necessary, even in small laboratories [3]. The miniaturization of the separation process has a number of advantages, which include low reagent consumption, integration of a series of analytical steps, rapid analysis and relative low unit cost but require to detect and measure
∗ Corresponding author. Tel.: +39 02 2399 3700; fax: +39 02 2367 604. E-mail address: [email protected] (I. Rech). 0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2009.09.058
the fluorescence from smaller and smaller samples, with ultraweak intensity, down to a few photons per second. High sensitivity detection systems in ultracompact apparatus are thus required. The state of the art [4–6] for MCE-based DNA analysis instruments makes use of photomultiplier tubes (PMTs) or highsensitivity CCDs. Very high PMT internal gain (105 ) produces high output pulses and makes possible to detect even single photons; their wide sensitive area (diameters from 10 mm to various centimeters) simplifies the design of the optical systems. However, PMTs present some drawbacks that prevent their use in compact genotyping systems: they cannot be integrated, so detection of the four bases can occur only using four different PMTs, increasing size and cost. Finally, PMTs have lower quantum efficiency compared to solid-state detectors, especially in the red and near-infrared spectrum. In recent years high-sensitivity CCDs have been employed [7]. They present very high quantum efficiency and, cooled to very low temperatures, very low noise as well [8]. Such low temperatures (about −50 ◦ C) are obtained using air or a liquid coolant, resulting in a bigger and more delicate apparatus. In summary, even if CCDs are commonly used in DNA sequencing, they are not the best solution regarding simplicity, cost and dimensions. The ideal detection head for low cost genotyping systems for large scale distribution should consist of only 4 pixels, one for each wavelength, capable of detecting single photons to properly detect even short phenomena, lasting for a few tens of milliseconds. For CCDs this is generally
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voltage. The bias voltage is then restored in order to detect another photon. This operation requires a suitable circuit that is usually referred to as quenching circuit [10,11]. SPADs have good quantum efficiency, better than PMTs, and all the advantages of solid-state devices (miniaturization, ruggedness, low voltage, low power, and reduced cost). Therefore a monolithic array of SPAD detectors could represent a breakthrough for the evolution of genotyping systems, because it allows to combine very high sensitivity with extreme compactness and simple system architecture, thanks to the low number of pixels to manage and the moderate cooling (−15 ◦ C) necessary for the best performances. The whole optical detection head, including the array detector and the associated electronics are described in Section 2. The structure and the fabrication process of the array detector are described in Section 2.3, while in Section 3 the system performances are reported. 2. Materials and methods
Fig. 1. LIF-MCE apparatus for DNA analysis; (a) scheme of the proposed apparatus, (b) photograph of the apparatus.
LIF-MCE genotyping systems are based on chain termination method [12] developed by Frederick Sanger in 1977. This method first involves breaking chromosomes, which range in size from 50 million to 250 million bases, into much shorter pieces. Each of this is used as a template to generate a set of fragments that differ in length by a single base, that will be identified in a later step, thanks to the labeling of each dideoxynucleotide chain-terminators with a different fluorescent dye. The fragments in a set are separated by microchip electrophoresis, i.e. the movement of an electrically charged substance under the influence of an electric field. Due to their size, fragments have different frictional forces, so they will separate from one another as they migrate through a buffer solution. Identifying the final base at the end of each fragment, it’s possible to recreate the original sequence of As, Ts, Cs, and Gs for each short piece generated in the first step. Thus, a system for DNA sequencing or genotyping should include (1) an ultra-stable high voltage section to supply correct voltages for the electrophoretic separation, (2) one or more laser to excite fluorescence of the different markers, (3) an optical system that focuses laser light on separation channel, collects fluorescence signals and allows the distinction of the four different fluorescence wavelengths, and (4) a detection unit composed at least of four pixels, one for each marker, with very high sensitivity, down to few photons per second. Moreover, a movement unit is required to automatically align the capillary with the laser spot. In Fig. 1(a) is shown a scheme of the apparatus here proposed. The LIF module size is 19 cm (L), 12.5 cm (W), 15 cm (H) (Fig. 1(b)). 2.1. Fluorescence excitation and electrophoretic separation
difficult, since in short integration times the noise relative to single pixels is no longer due to thermal noise but to reading noise independent of integration time. In this paper we present a complete system based on a custom solid-state single photon avalanche diode (SPAD) array detector. These microelectronic devices join the advantage of PMT and CCD solution. A SPAD is essentially a p-n junction operated in Geigermode, i.e. biased at a voltage higher than the breakdown [9]. In this operating regime, a single carrier generated in the depletion region can trigger a self-sustaining avalanche current. The current swiftly rises with nanosecond or subnanosecond risetime to a macroscopic steady level in the milliampere range, which can be easily discriminated. If the primary carrier is photogenerated, the leading edge of the avalanche pulse marks the arrival time of the detected photon. After the avalanche is triggered, the current keeps flowing until the avalanche is quenched by lowering the bias below the breakdown
Markers considered for the project of the entire system are Beckman-Coulter biomarkers, that fluoresce at 670, 705, 775 and 810 nm (Fig. 2). The choice of these markers allows the use of two simple, very low-cost and compact laser diode (650 nm and 755 nm) to excite the fluorescence, instead of bulky and costly lasers needed for other types of markers (typically Argon lasers). As for the microchip for the electrophoretic separation of the fragments, a Micronit microchip is used, characterized by very small dimensions (45 mm × 15 mm × 1.8 mm) and a channel width of 50 m. 2.2. Optical system The key elements to achieve high sensitivity are optical system and detection unit. Optical system has to collect emitted light
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focusing fluorescence signal on the detector. Moreover, a dispersing section has to spatially separate fluorescence wavelengths. A detailed diagram of the optical module is shown in Fig. 3(a). The excitation beams from two laser diodes (650 nm and 755 nm) are coupled into a microscope objective with a high numerical aperture, which focuses the beams into a 50 m-spot within the sample. The emerging fluorescence is collected by the same objective and separated from the excitation beams by dichroic mirrors. For a better filtering of the source signals, a series of optical filters are placed after dichroic mirrors. The four remaining wavelengths are spatially separated by a transmission grating (600 l/mm Thorlabs) and then focalized on array detector with a collimating triplet (GLC002 by Melles Griot). The optical filters were provided by Chroma Technology USA. We used a laser diode TOLD 9442 manufactured by Toshiba, which provides up to 5 mW of optical power at 650 nm and a laser diode L7910 manufactured by Hamamatsu capable of an output power of 4 mW at 750 nm. Fig. 2. Markers’ emission spectrum.
2.3. Detection unit: single photon array detector As for the detection unit, it has to be able to detect four fluorescence signals with ultrahigh sensitivity and with short acquisition
Fig. 3. Optical system; (a) block diagram, (b) photograph of the custom optical breadboard.
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Fig. 6. Photon detection efficiency of the SPAD array: due to the good uniformity of the technology process PDE spread is limited to a few percent and the curves overlap.
Fig. 4. Optical simulation used for custom design of array detector; (a) optical simulation, (b) layout of custom design array detector.
time (about 100 ms), in order to give an electropherogram with a good temporal resolution. Since high-performance CCDs have best sensitivity only for long acquisition times and since detector has to collect light from only four spot simultaneously, a simpler and more efficient approach than image acquisition may be sought. Performing simulations of the entire optical system, it was possible to determine (Fig. 4(a)) the exact positions on image plane of the four wavelengths, spatially separated by the transmission grating. Moreover, simulation showed how a certain bandwidth in fluorescence emission spectrum translates in line-width on image plane, as a function of the central wavelength. Here we present a custom designed SPAD array with pitch between pixels and active area diameter specifically designed to efficiently detect Beckman markers’ fluorescence signal (Fig. 4(b)). The choice of active area dimension allows a spatial filtering of non-fluorescence wavelengths. In this work, array detector was designed so that each pixel detects only 10 nm-bandwidth around central fluorescence wavelength in order to reduce the crosstalk between different markers. Fig. 5 shows the cross-section of an individual SPAD element in the array. The double-epitaxy structure provides a low series
Fig. 5. Cross-section of a double-epitaxy SPAD.
resistance for the avalanche current. The p+ boron implantation defines the high field region, where avalanche multiplication takes place. Deep phosphorus diffusions provide full electrical isolation among SPAD elements [13]. A monolithic array of 4 SPAD detectors (Fig. 4(b)) was fabricated on 4 in. wafers at IMM-CNR (Bologna) by using the fabrication process described in ref. [13]. Suitable gettering processes were used to avoid the steep increase of the dark counting rate with the detector diameter that was observed in the previous generations of SPAD devices [14]. Each SPAD has an active area of 50 m × 70 m and the pitch between SPADs is designed to optimize fluorescence collection when Beckman Coulter biomarkers are used (Fig. 2): distances between adjacent couples of devices is 165, 327 and 191 m, respectively. Position of each SPAD determines central detection wavelength, while longitudinal width (70 m) corresponds to a wavelength bandwidth of 10 nm in the worst case. To assess the suitability of the available SPAD arrays to the production of a parallel detection system, fabricated devices were fully characterized. We first tested individually the detectors in the array in order to evaluate their performances; the crosstalk between different detectors was then measured to verify the characteristics of our devices when operated simultaneously. 2.3.1. Photon detection efficiency The photon detection efficiency (PDE) of the array was evaluated operating the SPADs at 5 V excess bias voltage. Its behavior as a function of wavelength is shown in Fig. 6. The efficiency has a peak of about 50% at 550 nm and is about 12% at 820 nm. These values are consistent with the structure of the photon absorption region, which is about 5 m thick. Different pixels in the array show the same shape of the efficiency function, with a spread limited to a few percent. These variations are due both to the spread of the detector overvoltage and to the process non-uniformities over the chip. 2.3.2. Dark counting rate Even without illumination, thermally generated electron–hole pairs can trigger an avalanche in the device, thus producing dark counts. The dark counting rate (DCR) is affected by a Poissonian fluctuation that represents the noise of the detector. Fig. 7 shows the dark counting rates, sorted in increasing order, of the four SPADs in a typical array. Fig. 8 shows the temperature behavior of the DCR for a single SPAD. As a general rule, dark counts are caused by the combined effect of band-to-band tunneling and Shockley Reed Hall generation; this two contributions show a different temperature behavior, with tunneling effect dominating at low temperatures and SRH generation at high temperatures. The electric field in the
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Fig. 7. Dark counting rate.
depletion region of the detector can be engineered to reduce the contribution of band-to-band tunneling, which limits the possibility of reducing the DCR by cooling the device. The curves in Fig. 8 are relative to two different design of the detector. Older devices (“standard” electric field) showed a pronounced tunnel effect that limited the working temperature to −5 ◦ C, because further reduction of the temperature did not reduce significantly the dark counts. The detectors used in our apparatus (“engineered” electric field) were specifically designed to cut the contribution of tunneling, so that the temperature dependence of the DCR is now much steeper [14,15]; its worth noting that, in respect to the older design, the dark count rate is lower even at room temperature. 2.3.3. Crosstalk Crosstalk represents the correlation between the output signals of different channels. Ideally the counting rates of two different detectors in the array should be totally uncorrelated. Unfortunately optical coupling between nearby devices causes some sort of correlation; at the device level this is due to the avalanches that are triggered in one detector as a result of the optical emission of another SPAD that is subject to current flow. Crosstalk effects were evaluated using the time correlated single photon counting (TCSPC) technique; measurements were performed at −10 ◦ C, operating the SPADs at 5 V excess bias voltage and with an holdoff time of 100 ns. Measures show that the crosstalk varies from 8.1 × 10−4 to 1.7 × 10−3 , depending on the distance between the considered detectors. The measured crosstalk does not follow a
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monotonic behavior: it first increases and then smoothly decreases in an unpredicted fashion. This behaviour can be explained considering indirect optical paths reecting off the bottom silicon–air interface where the classical total internal reection takes place for rays above the critical angle. All the photons emitted by a SPAD and reecting at the bottom of the chip with an angle of incidence around (or greater than) the critical angle can reach another device causing optical crosstalk. Conversely, only a fraction of the photons reecting with an angle of incidence smaller than the critical angle can reach another device; for this reason their contribution to the crosstalk is less significant. Since larger distances between SPADs imply greater angles of incidence for the reected paths going from one device to the other, the observed “bell shape” can be ascribed to the overcoming of the critical angle. In our labs it was developed an accurate 3D optical model that takes into account substrate doping level, silicon absorption lengths as a function of wavelength and isolation doping level, considering also secondary effects due to rejection on lateral surfaces [16]. 2.4. Optical alignment In currently used MCE setups, the operator is often requested to perform several manual operations every time the chip is removed, such as washing, refilling, etc. These operations include the alignment of the microchannel with the optical path of the LIF module. In order to avoid a time-consuming manual alignment, we have developed a fully automated procedure. This procedure is based on the measurement of the faint signal generated by a wide-spectrum led light that is scattered from the glass-microchannel discontinuity and is not completely cut-off by the optical filter system. By using a micrometric translation stage, the laser beam is scanned onto the electrophoresis chip over a line perpendicular to the microchannel. An onboard microcontroller moves step-by-step to the position of the translation stage, counts the photon pulses from a SPAD detector at each position, and sends the recorded counts to the PC through a USB interface. The scattered light signal attains its maximum when the microchannel is perfectly aligned to the optical path. The remote PC performs all the computations needed to find out this maximum; then it communicates the result to the microcontroller, which finally aligns the system to the position of the maximum. It is worth stressing that it is the precise alignment guaranteed by the automated system that makes possible to perform the experiments with high repeatability and practically zero failure rate. In fact, since the laser spot diameter and the microchannel section have the same size (approximately 50 m), even a small misalignment (few microns) between them causes a significant reduction of the instrument sensitivity. Due to the large depth of field of the optics, the position along the z-axis is simply adjusted via a vertical translator before carrying out a series of measurements. 2.5. Electronic section and software interface
Fig. 8. Noise temperature behavior of the noise of two different generations of SPAD devices.
To implement the pinched mode injection-separation sequence, three independent high-voltage power supplies are developed using ultra-compact DC-HV DC converter from EMCO. A closed loop system was designed to obtain very stable and precise voltage levels. The maximum output voltage is 4 kV. The currents are monitored during the DNA injection and separation; the available measurement ranges are 0–25, 0–50, and 0–100 A. Platinum electrodes are connected to the microchip reservoirs by means of a servo-mechanical actuator. Chip holder, plexiglass electrode seat and connection system are designed to obtain a very compact system. Each parameter in the apparatus is fully controlled by a PC. The adjustable parameters include the working conditions of the SPAD
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structure allowed a very simple testing of all the parts, but implies a sizable encumbrance of the optical system. To overcome this problem, a different structure has been realized. The apparatus here described is based on a transmission grating as separation element and two optical groups to collect fluorescence light and to focus it on the SPAD array. Assuming that the fluorescence from the microcapillary acts like a Lambertian source it can be note that the power radiation in terms of W/m2 ·sterad is: B(˛) = B(0) · cos(˛) So, the power emitted from the source on a solid angle d is: d = AS · B(˛) · d˝ = AS · B(0) · cos(˛) · d˝ where d and d␣ are related by d˝ = 2 · sin(˛) · d˛ The collection efficiency of the optical system is given by the ratio between incident light with an angle lower than acceptance angle and the total light emitted:
˛max
=
0
/2 0
Fig. 9. Optical dispersion of the realized apparatus.
detectors (bias voltage, dead-time, temperature), and the output power of the excitation laser. It is also possible to perform a series of runs while varying step-by-step a particular parameter, e.g., the laser power or the injection voltage. The instrument is connected to a nearby PC through a USB interface. 3. Results and discussion Optical system and detection unit are the key blocks in the apparatus to achieve high sensitivity. As for the optical system, one of the most important parameters is the true optical dispersion, because this characteristic determines the ability to distinguish and spatially filter different wavelengths. Fig. 9 shows the good agreement between experimental data and optical simulation. To obtain the experimental data, we collected the light coming from transmission grating with a focusing lens GLC002 (Melles Griot). Image on focal plane was acquired with a CCD camera. Spots corresponding to wavelengths with 10 nm bandwidth were realized using an Oriel monochromator. The huge potential of SPAD detector in LIF-MCE DNA analysis was demonstrated in a previous work of our group [17] in which the use of two SPADs as detection head allowed a sensitivity limit of 3 pM with 50 pl injection volume. The apparatus here presented represents the natural evolution of the previous one in terms of compactness and type of analysis, without decreasing the overall performances. In fact, since the optical collection efficiency of the two systems is the same, the apparatus presented is able to perform DNA sequencing and genotyping analysis, with analogous detection limit in extremely compact dimensions. The apparatus described in [17] employed interferential filters to separate fluorescence wavelengths and optical fibers to couple fluorescence light on the corresponding SPAD detector. For that system its possible to derive a peak collection efficiency of 1.6%, considering fluorescence emission from the microcapillary as a lambertian source and accounting for filter’s transmittivity, light fiber coupling and numerical aperture of the collection lens. This
2 · sin(˛) · AS · B(0) · cos(˛) · d˛ 2 · sin(˛) · AS · B(0) · cos(˛) · d˛
=
NA 2 n
where NA = n·sin(˛) is the numerical aperture and n is the refractive index of the material. So the overall optical efficiency, considering the transmission grating transmittivity is given by: = · = 0.16 · 0.35 = 5.6% This value is confirmed by experimental results obtained using an optical fiber as source. This is a good choice to emulate a capillary for LIF-MCE: 50 micron core dimension fit with typical cross-section of microchip capillary and its possible to generate test wavelength with a monochromator in all visible range, covering most of possible marker’s fluorescence wavelengths. In the next months we will perform a full set of DNA analysis to validate our apparatus, but since the system here presented and the apparatus described in [17] have comparable optical efficiency, we are confident that even the detection limit will be comparable. 4. Concluding remarks The development of an ultra-compact instrument based on single photon avalanche diode (SPAD) and able to perform MCE DNA sequencing analysis is here reported. The planar epitaxial SPAD combine the typical advantages of microelectronic devices (miniaturization, ruggedness, low voltage, low power, low cost, etc.) with high sensitivity. The suitability of such SPAD to MCE has been demonstrated in recent works. The apparatus presented here could represent a breakthrough in the development of genetic diagnostic system thanks to the extreme compactness and the low cost of basic components. At the best of our knowledge, it is the most compact system for genotyping reported in literature and it could open the way to the development of a new category of genotyping systems, portable and low-cost. The intrinsic performance of the system was tested demonstrating a good fitting with simulations. A comparison with other assays will be accomplished in the next months. Since this system is the natural evolution of a former work (supported by the Italian Ministry of University and Research within the framework of the Project FIRB No. RBNE01SLRJ\ 001 “Microsystems for genetic diagnostics”) that brought to a DNA separation system with sensitivity better
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than 3 pM, we are confident that the system will show a sensitivity comparable with the best DNA sequencers. References [1] E. Verpoorte, Microfluidic chips for clinical and forensic analysis, Electrophoresis 23 (2002) 677–712. [2] N.J. Dovichi, J. Zhang, K.O. Voss, D.F. Shaw, et al., A multiple-capillary electrophoresis system for small-scale DNA sequencing and analysis, Nucleic Acids Research 27 (1999) 24. [3] T. Vo-Dinh, B. Cullum, Biosensors and biochips: advances in biological and medical diagnostics, Fresenius Journal of Analytical Chemistry 366 (6–7) (2000) 540–551. [4] Q. Gao, Y. Shi, S. Liu, Multiple-channel microchips for high-throughput DNA analysis by capillary electrophoresis, Fresenius Journal of Analytical Chemistry 371 (2001) 137–145. [5] L. Alaverdian, S. Alaverdian, O. Bilenko, I. Bogdanov, E. Filippova, D. Gavrilov, B. Gorbovitski, M. Gouzman, G. Gudkov, S. Domratchev, O. Kosobokova, N. Lifshitz, S. Luryi, V. Ruskovoloshin, A. Stepoukhovitch, M. Tcherevishnick, G. Tyshko, V. Gorfinkel, A family of novel DNA sequencing instruments based on singlephoton detection, Electrophoresis 23 (16) (2002) 2804–2817. [6] A. Stepukhovich, A. Tsupryk, O. Kosobokova, D.N. Gavrilov, B. Gorbovitski, G. Gudkov, G. Tyshko, M. Tcherevishnik, V. Gorfinkel, Analysis of DNA sequencing systems based on capillary electrophoresis, Technical Physics 53 (6) (2008) 763–775. [7] D. Meldrum, Automation for genomics, part two: sequencers, microarrays, and future trends, Genome Research 10 (2000) 1288–1303. [8] J.R. Janesick, Scientific Charge-Coupled Devices, SPIE Press Monograph, 2001. [9] M. Ghioni, A. Gulinatti, I. Rech, F. Zappa, et al., Progress in silicon single-photon avalanche diodes, IEEE Journal of Selected Topics in Quantum Electronics 13 (4) (2007) 852–862. [10] S. Cova, M. Ghioni, A. Lacaita, C. Samori, et al., Avalanche photodiodes and quenching circuits for single photon detection, Applied Optics 35 (1996) 1956. [11] A. Gallivanoni, I. Rech, D. Resnati, M. Ghioni, et al., Monolithic active quenching and picosecond timing circuit suitable for large–area single–photon avalanche diodes, Optics Express 14 (12) (2006) 5021–5030. [12] F. Sanger, S. Nicklen, A.R. Coulson, DNA sequencing with chain-terminating inhibitors, Proceedings of the National Academy of Sciences of the United States of America 74 (12) (1977) 5463–5467. [13] A. Lacaita, M. Ghioni, S. Cova, Double epitaxy improves single-photon avalanche diode performance, Electron. Lett. 25 (1989) 841–843. [14] M. Ghioni, A. Gulinatti, P. Maccagnani, I. Rech, et al., Planar silicon spads with 200 (m diameter and 35 ps photon timing, in: Proceedings of SPIE, Optics East 2006, Advanced Photon Counting Techniques 6372, Boston, MA, USA, 2006. [15] M. Ghioni, A. Gulinatti, I. Rech, P. Maccagnani, et al., Large-area low-jitter silicon single photon avalanche diodes, paper presented at SPIE Photonics West 2008—Quantum Sensing and Nanophotonic Devices V, San Jose, CA, USA, January 19–24, 2008. [16] I. Rech, A. Ingargiola, R. Spinelli, I. Labanca, et al., Optical crosstalk in single photon avalanche diode arrays: a new complete model, Optic Express 16 (12) (2008) 8381–8394. [17] I. Rech, S. Cova, A. Restelli, M. Ghioni et al., Microchips and single-photon avalanche diodes for DNA separation with high sensitivity, Electrophoresis 27 (19) (2006).
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Biographies Ivan Rech, born in Milan in 1976, graduated in 2000 in Electronic Engineer at Politecnico di Milano, where he is currently Assistant Professor. PhD in Information Technology at Politecnico di Milano in 2004. His current research interests are focused on the development of single photon detectors and associated electronics for new microanalytical techniques in biomedical, genetic and diagnostic applications. He developed a photon detection module with high timing resolution, which has been employed in florescence measurements on single molecules at the Department of Chemical Biology, Harvard University. He was involved in several interdisciplinary research projects on molecular biology, quantum cryptography, adaptive optics in astronomy, developing dedicated instrumentation and electronic devices. Stefano Marangoni is Ph.D. Student in Information Technology at the Politecnico di Milano, Italy. He has been working on the development of ultra-compact electronic systems for very faint luminescence analysis with single photon photodetectors. His work is focused on complete SPAD array-based systems, ranging from power electronic for CE separation processes and digital programmable electronic development to conjugated optical system project and in-depth analysis of optical crosstalk in detector arrays. Angelo Gulinatti was born in Codogno, Italy, in 1977. He received the Laurea degree in electronic engineering and the Ph.D. degree in information technology (both summa cum laude) from Politecnico di Milano, Milan, Italy in 2003 and 2007, respectively. Since 2008 he is Assistant Professor at Politecnico di Milano where he carries out his research activity in the development and modeling of silicon single photon avalanche diodes (SPAD). Massimo Ghioni (M’91) received the Laurea degree (cum laude) in Nuclear Engineering from the Politecnico di Milano, Italy in 1987. Since 1990, he has been with the Department of Electronics and Information of the Politecnico di Milano, where he is currently Full Professor of Electronics. In 1992 he was visiting scientist at the IBM T.J. Watson Research Center, Yorktown Heights, NY, USA, where he developed a new CMOS compatible SOI photodetector for optical datacom applications. His current research interests are focused on the development of SPAD detectors and associated electronics for applications in physics, astronomy, and molecular biology. He is author of over eighty papers in international refereed journals and conferences and of six U.S. and European patents. Dr. Ghioni received the AEI (Italian Association of Electrical and Electronic Engineers) Award in 1996 and the IEEE PELS Transactions Prize Paper Award for 2004. Sergio Cova, born in 1938 Roma, Italy, he received a doctor degree in Nuclear Engineering in 1962 from Politecnico di Milano, Italy, where he is Full professor of Electronics since 1976. Fellow of the IEEE, he is author of over a 170 papers in international refereed journals and conferences and of five international patents (USA and Europe). He has given innovative contributions in the research and development of detectors for optical and ionising radiations and associated electronics, of microelectronic devices and circuits, of electronic and optoelectronic measurement instrumentation and systems. He pioneered the development of single photon avalanche diodes SPAD, inventing the active-quenching circuit AQC, which opened the way to their application, and devising new SPAD device structures. He collaborated to interdisciplinary research in physics, astronomy, cytology and molecular biology, developing dedicated electronic and optoelectronic devices and instrumentation.