- Email: [email protected]
Extremely integrated device for high sensitive quantitative biosensing
Sensors and Actuators B 209 (2015) 1011–1014
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Short Communication
Extremely integrated device for high sensitive quantitative biosensing Lucio Renna ∗ , Clelia Galati, Natalia Spinella, Massimo Mazzillo, Salvatore Abbisso, Piero Giorgio Fallica STMicroelectronics, Stradale Primosole, 95121 Catania, Italy
a r t i c l e
i n f o
Article history: Received 25 August 2014 Received in revised form 3 December 2014 Accepted 4 December 2014 Available online 12 December 2014 Keywords: Proteins sensor Luminescence photodetector SiPM
a b s t r a c t The development of integrated sensors is a challenge currently facing biodiagnostics where multiplexing and high sensitivity detection are frequently required. This work follows an approach based on silicon photomultiplier detection, phosphorescent nanobeads and compact front-face light-source configuration. The advantages of this approach also derive from the sensitivity, compactness, robustness and low operating voltage of SiPM devices. An experiment was performed to demonstrate performance in terms of sensitivity, with the results allowing the estimation of a detection limit for proteins of 1–10 pg/mL. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The development in miniaturized devices for biodiagnostics will soon spark the diffusion of high performance healthcare microsystems, rendering the so-called “point-of-care” devices a reality [1,2]. Fluorescence based microarrays for DNA sequencing and protein recognition tests are finding widespread application, with several advantages with respect to conventional ELISA tests such as reduced reagent consumption and multiplexing [3–5]. High sensitivity is often required for the implementation of such devices in various clinical tests [6–8] and various strategies have been applied to improve performance, including the use of very efficient quantum dots as fluorophores [9], increasing the surface loading capacity [10] and fluorescence amplification techniques [11,12]. The merging of microarrays with microfluidics has also been the subject of intensive research [13] due to significant advantages associated with reducing manual operations and expensive reagent consumption, as well as allowing much more rapid detection [14–16]. In order to obtain the desired sensitivity, fluorescence detection of microarrays is typically performed with confocal microscopes or relatively bulky readers equipped with laser sources and photomultipliers [17].
∗ Corresponding author. Tel.: +39 0957407442. E-mail address: [email protected] (L. Renna). http://dx.doi.org/10.1016/j.snb.2014.12.014 0925-4005/© 2014 Elsevier B.V. All rights reserved.
The Silicon Photomultiplier (SiPM) is a new type of photoncounting device consisting of a matrix of Geiger-mode Avalanche Photodiodes (GMAPs) with resistive quenching, connected in parallel to a single readout element (see Supplementary Information). The SiPM allows counting of the number of photons interacting with the sensor up to a saturation value that depends on the total number of photodiodes in the array [18,19]. There is clear evidence that the SiPM will progressively replace standard vacuum Photo-Multipliers Tubes (PMTs) in a wide range of applications, including high energy physics, nuclear medical imaging and recently biophotonics, due to its very fast timing response, high photon detection efficiency (PDE) in all the visible range, low operating bias, insensitivity to magnetic fields and compactness [20]. Others solid state detectors could also be considered, like CMOS photodiodes, Avalanche photodiodes, etc [21–25]. A straightforward comparison of the efficiency of different systems is not always possible because of the varying targeted reactions and biomarkers (glucose, lactate, cells, etc) and their typical concentrations in the analytical sample, as well as the different methods (chemiluminescence, fluorescence, electroluminescence, etc) and purposes (e.g. wearable or disposable device or laboratory instruments) involved. In [24], for example, which discusses an ELISA test with very good sensitivity, a CCD camera cooled at −45 ◦ C is used in combination with chemiluminescence and immobilization of antibodies in capillary tubes. In this project, the SiPM was chosen as it suits the typical requirements of an integrated lab on a chip device for biodiagnostics: good sensitivity, low operating voltage, fast response,
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Fig. 2. Oscilloscope waveform acquisition on a 600 m × 600 m (10 × 10 cells) SiPM device in dark condition.
Fig. 1. Prototype device (approx. 2 × 4 cm) with bonded SiPM test pattern.
minimum or no external amplification electronics, and cost [26]. Compatibility with conventional microarray technology and the possibility of exploiting parallel production technology were also determinant factors. The main drawback of SPAD and SiPM devices is the relatively high dark count rate, often the limiting parameter for the limit of detection (LoD) of a particular experiment, as in the case of chemiluminescence [5,25]. As discussed later in the paper, however, the typical dark count rate is not the limiting aspect in this case due to the peculiarity of the adopted approach. Other artifacts, on the other hand, need to be faced. The following report discusses a novel SiPM-based analyzer (SiPMAn) device for the implementation of integrated fluorescence-based microarrays. The main components of the devices are depicted in Fig. 1. The method employed can be described thus: if we set a biodiagnostic test (e.g. immunoassay) to occur onto the outermost surface of the sensor, then the final step of such test can be arranged (as usual in microarray technology) to bind luminescent reporters to the surface. This work involves the use of platinum-polystyrene, chelates-based, phosphorescent nanobeads with decay times in the order of microseconds as labeling strategies (see Supplementary Information). The reporters on the sensor surface are excited by means of a pulsed light, with pulses much shorter than the fluorescence decay. The fluorescence signal, which is proportional to the number of fluorescent nanobeads present on the surface, can then be read by the sensor during the light-off period. A model experiment was performed with the aim of evaluating the performance in terms of the sensitivity and dynamic range of such a device. 2. Experimental details A squared 600 m × 600 m SiPM (10 × 10 cell array) was electrically accessed by wire bonding onto a suitable board, closed in a black and dark box and connected to an external power supply driving the SiPM at 3 V overvoltage with respect to the breakdown (see also Supplementary Information). No amplification stage was applied on the device output. The device reading output was then connected to a high band pass oscilloscope able to easily distinguish the single cascade events. Fig. 2 shows a single 200 s oscilloscope track, acquired on a polarized device with no light exposure, featuring a constant, slightly noisy baseline with a few clearly distinguishable narrow 5 mV (approx. 2 ns) peaks, indicating single, random avalanche cascade events.
The pulsed laser source was brought into the dark box to strike the SiPM surface via an optical fiber. The laser pulse duration was tuned by the laser driver to 2 ns. The laser pulse and a 200 s oscilloscope acquisition track were triggered by a pulse generator. A 2-s duty cycle was set to allow all the fluorescent molecules to return to their fundamental state, to avoid artifacts like steady state behavior. After preliminary device investigation regarding dark noise, some devices were selected to have a low and comparable dark count rate, and were subjected to surface spotting with 16 (4 × 4) drops of a 330 pL luminescent nanobead solution. Drop spotting was performed through the precise tuning and alignment of a piezo-spotter. Three scaled order of magnitude concentrations, from 2 × 1011 to 2 × 1013 beads/mL, were spotted to test both device sensitivity and dynamic range. These concentrations were chosen to match the typical sensitive immunoassay requirements, as reported in the final results. After final tuning of the distance between the optic fiber output and the device surface, the oscilloscope tracks for the devices were acquired and stored. The oscilloscope sampling rate was set to 10 GSample/s, which was high enough for good-quality acquisition of the single 2 ns peak events. A single 200 s track therefore includes 2 × 106 data points. 3. Results and discussion Fig. 3 depicts two 200 s tracks, acquired on the reference sample (no fluorescent molecules on the SiPM surface) and on the 2 × 1013 beads/mL spotted sample. In Fig. 3a, two main differences are evident with respect to the tracks in Fig. 2, where no laser pulse was applied: • The presence of a saturation peak at the beginning of the tracks. This peak is due to simultaneous photon detection of all the 100 SiPM cells because of the very high number (about 3 × 108 ) of the incoming photons during the 2 ns laser pulse. • A much higher number of dark events following the strong initial peak, where no light is more frequent. These secondary counts (artifacts due to the afterpulsing phenomena [27]) therefore represent the real background against which the signals have to be compared. By comparing the two waveforms, the much higher signal (crowded and piled-up detection events) in the bead containing sample is clearly evident in comparison with the reference sample. The quantitative information was accessed by acquiring 21 tracks for each sample and implementing a data elaboration protocol. A suitable algorithm was developed for the offline data processing, including waveform processing consisting of detecting
L. Renna et al. / Sensors and Actuators B 209 (2015) 1011–1014
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The limit of detection can be determined via extrapolation of the concentration, generating a net signal 3 times higher than the mean standard deviation of the reference sample [28,29]. The resulting LOD is of 3.8 × 105 fluorescent nanobeads. In terms of sensitivity for a generic immunoassay, we can consider this quantity of fluorescent beads to have bonded to the active sensor surface as a consequence of the last incubation step of a microarray immunoassay. Referring to a generic immunoassay, one bead relates to the recognition of one protein on the surface. Depending on the specific biomarker molecular weight (e.g. in the 10–100 kD range) and considering the analysis volume of about 10 L usually involved in microarray applications, then the beads detection limit can be converted into a concentration LoD in a range of 1–10 pg/mL. Such sensitivity is high enough to detect important biomarkers like those related to Alzheimer disease [6], allergy detection [8] and thyroid monitoring [7].
4. Conclusions and outlook
Fig. 3. Acquired raw waveform on 10 × 10 cells, SiPM devices, subjected to 2 ns laser pulse excitation, with: (a) no nanobeads dispensed (reference sample) and (b) with nanobeads dispensed (16 drops at 2 × 1013 beads/mL). The starting laser pulse is evidenced by the intense saturated SiPM signal.
each peak against the background and subsequently integrating and summing them over a specific measurement period. Integration operations commence after the first 2 s to remove the bias of the strong after-pulsing in the first part of the curves, where the SiPM signal remains too close to the saturation value. Data analysis details are reported in supplementary information. Fig. 4 shows the results of data processing, where the net signal values (averaged signal minus averaged signal of the reference sample) and mean standard deviation over the 21 tracks are plotted. The inset shows a magnified area of the plot close to the zero signal. A reasonably dynamic range is evidenced in the scanned concentrations, although some deviation from the linearity is observed.
We have developed a novel method for biodiagnostic applications: using the active region of silicon photomultipliers as sensing areas bestows devices with several characteristics that are highly suited to the requirements of point-of-care systems, including the high integration level, low operating voltage, quantitative information with high sensitivity and good dynamic range. The system is considered compatible with the high throughput microarray technology. Microarrays of differing primary probes can be hosted on a corresponding array of micrometric SiPM detectors. Typical Integrated Circuit technology and chemical functionalization steps can be implemented to control the outermost layer chemistry to obtain the desired properties, such as binding capacity and reduction of artifact signals due to nonspecific interactions [30]. After functionalization, the SiPM surface can be precisely spotted with primary antibodies by means of conventional robotized equipment. The proposed approach is however compatible with different dispensing techniques of possible future implementation [31]. Although an oscilloscope and laser equipment was used for the above characterization, the long luminescence relaxation time and the mV range SiPM readout peaks will allow for future miniaturized apparatus developments based on LED excitation and simple reading electronics. The structure will also allow microfluidics to be easily implemented to obtain fast reactions and low reagent consumption. Finally, the device structure can be based on inexpensive elements since no expensive or bulky optical components (e.g. filters) are required for the fluorescence reading. Most of the construction steps can be implemented with the widest parallel production technology: Integrated Circuits. In the near future, we shall develop SiPM devices with smaller cells. Subsequent experiments can therefore be performed on devices smaller than 150 m × 150 m, which matches the characteristics of typical microarray spot dimensions more closely. Reduction of afterpulsing background is also being researched to obtain still higher sensitivity. Work for the implementation of immunoassay for the detection of Alzheimer’s disease using SiPMAn is currently being researched in our laboratory.
Acknowledgements Fig. 4. Plot of processed waveforms results to obtain integral photocurrent value for concentrations of nanobead solutions varying across 3 orders of magnitude and for the reference sample. The inset on the right shows a detail close to the reference signal to extract the LOD.
We are grateful to R&D Pilot Line of STMicroelectronics for manufacturing the silicon photomultiplier devices. The Italian MiUR program FIRB-MERIT is acknowledged as the founder.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2014.12.014.
[29] V. Thomsen, D. Schatzlein, D. Mercuro, Spectroscopy 18 (12) (2003) 112. [30] M. Salim, S.L. McArthur, S. Vaidyanathan, P.C. Wright, Mol. BioSyst. 7 (2011) 101. [31] C. Zheng, J. Wang, Y. Pang, J. Wang, W. Li, Z. Ge, Y. Huang, Lab Chip 12 (2012) 2487.
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Lucio Renna received the M.S. degree in Chemistry and the Ph.D in Material Science from University of Catania, in 1999 and 2007 respectively. Since 1999 he joined STMicroelectronics, where he currently works in R&D department. During his experience in STMicroelectronics, he gained multidisciplinary competencies in the development of integrated devices for bio-diagnostic applications and since 2011 is member of technical staff at STMicroelectronics. His research activity is documented by more than 40 publications and patents. Clelia Galati graduated in chemistry with honors at the University of Catania in 1999. In the same year she received a fellowship for research activity at STMicroelectronics. In 2000, he joined STMicroelectronics, where she currently works in R&D department. She performed activity as researcher in the field of molecular electronics and chemical surface functionalization. Her current activity is mainly focused on development of biosensors, in health and environment field, and as supervisor for Research Unit STMicroelectronics in the frame of national projects. Her research activity is documented by more than 40 scientific papers and patents. Natalia Spinella graduated in Chemistry with honors at the University of Catania in 2000. During the first year of her degree she started an ongoing collaboration with the University of Catania, working with the chemical physic research group. Here, she has performed research activities focused on the surface characterization of polymeric, biological and inorganic materials by time-of-flight secondary ion mass spectrometry analysis. In 2004, she joined STMicroelectronics, where she currently works as researcher in the field of chemical and biological sensor. Her research activity is documented by more than 10 publications and patents. Massimo Mazzillo received the M.S. degree with honors in physics from the University of Bari, in 2002. Since 2002, he has been with R&D STMicroelectronics, Catania, as optical sensors designer. Most of his research activity has been focused on the development of silicon single-photon detectors for biomedical applications and high-energy physics experiments and silicon carbide photodiodes for ultraviolet light detection. He is author of 14 patents and more than 80 papers published in international journals and conference proceedings. He serves as a Referee for the leading journals in the field of solid-state optical detectors and is member of STMicroelectronics Technical Staff. Salvatore Abbisso received the M.S. degree in electronics engineering from the University of Catania, Italy, in 2000. Since 2000, he has been with R&D, IMS, STMicroelectronics, Catania. Piero Giorgio Fallica is the Manager of “Advanced Sensors Development Group” inside R&D department of STMicroelectronics in Catania (Italy). He has received the degree in Physics from the University of Catania, Italy, in 1978. In 1981 he joined STMicroelectronics. He has more than 30 years’ experience in ICs, power electronics and sensors design. Starting from 1988, he managed the development of silicon sensors, mainly nuclear particle detectors and photo-sensors. International Patents issued: 11. Scientific papers and contributions to conferences: more than one hundred.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Short Communication
Extremely integrated device for high sensitive quantitative biosensing Lucio Renna ∗ , Clelia Galati, Natalia Spinella, Massimo Mazzillo, Salvatore Abbisso, Piero Giorgio Fallica STMicroelectronics, Stradale Primosole, 95121 Catania, Italy
a r t i c l e
i n f o
Article history: Received 25 August 2014 Received in revised form 3 December 2014 Accepted 4 December 2014 Available online 12 December 2014 Keywords: Proteins sensor Luminescence photodetector SiPM
a b s t r a c t The development of integrated sensors is a challenge currently facing biodiagnostics where multiplexing and high sensitivity detection are frequently required. This work follows an approach based on silicon photomultiplier detection, phosphorescent nanobeads and compact front-face light-source configuration. The advantages of this approach also derive from the sensitivity, compactness, robustness and low operating voltage of SiPM devices. An experiment was performed to demonstrate performance in terms of sensitivity, with the results allowing the estimation of a detection limit for proteins of 1–10 pg/mL. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The development in miniaturized devices for biodiagnostics will soon spark the diffusion of high performance healthcare microsystems, rendering the so-called “point-of-care” devices a reality [1,2]. Fluorescence based microarrays for DNA sequencing and protein recognition tests are finding widespread application, with several advantages with respect to conventional ELISA tests such as reduced reagent consumption and multiplexing [3–5]. High sensitivity is often required for the implementation of such devices in various clinical tests [6–8] and various strategies have been applied to improve performance, including the use of very efficient quantum dots as fluorophores [9], increasing the surface loading capacity [10] and fluorescence amplification techniques [11,12]. The merging of microarrays with microfluidics has also been the subject of intensive research [13] due to significant advantages associated with reducing manual operations and expensive reagent consumption, as well as allowing much more rapid detection [14–16]. In order to obtain the desired sensitivity, fluorescence detection of microarrays is typically performed with confocal microscopes or relatively bulky readers equipped with laser sources and photomultipliers [17].
∗ Corresponding author. Tel.: +39 0957407442. E-mail address: [email protected] (L. Renna). http://dx.doi.org/10.1016/j.snb.2014.12.014 0925-4005/© 2014 Elsevier B.V. All rights reserved.
The Silicon Photomultiplier (SiPM) is a new type of photoncounting device consisting of a matrix of Geiger-mode Avalanche Photodiodes (GMAPs) with resistive quenching, connected in parallel to a single readout element (see Supplementary Information). The SiPM allows counting of the number of photons interacting with the sensor up to a saturation value that depends on the total number of photodiodes in the array [18,19]. There is clear evidence that the SiPM will progressively replace standard vacuum Photo-Multipliers Tubes (PMTs) in a wide range of applications, including high energy physics, nuclear medical imaging and recently biophotonics, due to its very fast timing response, high photon detection efficiency (PDE) in all the visible range, low operating bias, insensitivity to magnetic fields and compactness [20]. Others solid state detectors could also be considered, like CMOS photodiodes, Avalanche photodiodes, etc [21–25]. A straightforward comparison of the efficiency of different systems is not always possible because of the varying targeted reactions and biomarkers (glucose, lactate, cells, etc) and their typical concentrations in the analytical sample, as well as the different methods (chemiluminescence, fluorescence, electroluminescence, etc) and purposes (e.g. wearable or disposable device or laboratory instruments) involved. In [24], for example, which discusses an ELISA test with very good sensitivity, a CCD camera cooled at −45 ◦ C is used in combination with chemiluminescence and immobilization of antibodies in capillary tubes. In this project, the SiPM was chosen as it suits the typical requirements of an integrated lab on a chip device for biodiagnostics: good sensitivity, low operating voltage, fast response,
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L. Renna et al. / Sensors and Actuators B 209 (2015) 1011–1014
Fig. 2. Oscilloscope waveform acquisition on a 600 m × 600 m (10 × 10 cells) SiPM device in dark condition.
Fig. 1. Prototype device (approx. 2 × 4 cm) with bonded SiPM test pattern.
minimum or no external amplification electronics, and cost [26]. Compatibility with conventional microarray technology and the possibility of exploiting parallel production technology were also determinant factors. The main drawback of SPAD and SiPM devices is the relatively high dark count rate, often the limiting parameter for the limit of detection (LoD) of a particular experiment, as in the case of chemiluminescence [5,25]. As discussed later in the paper, however, the typical dark count rate is not the limiting aspect in this case due to the peculiarity of the adopted approach. Other artifacts, on the other hand, need to be faced. The following report discusses a novel SiPM-based analyzer (SiPMAn) device for the implementation of integrated fluorescence-based microarrays. The main components of the devices are depicted in Fig. 1. The method employed can be described thus: if we set a biodiagnostic test (e.g. immunoassay) to occur onto the outermost surface of the sensor, then the final step of such test can be arranged (as usual in microarray technology) to bind luminescent reporters to the surface. This work involves the use of platinum-polystyrene, chelates-based, phosphorescent nanobeads with decay times in the order of microseconds as labeling strategies (see Supplementary Information). The reporters on the sensor surface are excited by means of a pulsed light, with pulses much shorter than the fluorescence decay. The fluorescence signal, which is proportional to the number of fluorescent nanobeads present on the surface, can then be read by the sensor during the light-off period. A model experiment was performed with the aim of evaluating the performance in terms of the sensitivity and dynamic range of such a device. 2. Experimental details A squared 600 m × 600 m SiPM (10 × 10 cell array) was electrically accessed by wire bonding onto a suitable board, closed in a black and dark box and connected to an external power supply driving the SiPM at 3 V overvoltage with respect to the breakdown (see also Supplementary Information). No amplification stage was applied on the device output. The device reading output was then connected to a high band pass oscilloscope able to easily distinguish the single cascade events. Fig. 2 shows a single 200 s oscilloscope track, acquired on a polarized device with no light exposure, featuring a constant, slightly noisy baseline with a few clearly distinguishable narrow 5 mV (approx. 2 ns) peaks, indicating single, random avalanche cascade events.
The pulsed laser source was brought into the dark box to strike the SiPM surface via an optical fiber. The laser pulse duration was tuned by the laser driver to 2 ns. The laser pulse and a 200 s oscilloscope acquisition track were triggered by a pulse generator. A 2-s duty cycle was set to allow all the fluorescent molecules to return to their fundamental state, to avoid artifacts like steady state behavior. After preliminary device investigation regarding dark noise, some devices were selected to have a low and comparable dark count rate, and were subjected to surface spotting with 16 (4 × 4) drops of a 330 pL luminescent nanobead solution. Drop spotting was performed through the precise tuning and alignment of a piezo-spotter. Three scaled order of magnitude concentrations, from 2 × 1011 to 2 × 1013 beads/mL, were spotted to test both device sensitivity and dynamic range. These concentrations were chosen to match the typical sensitive immunoassay requirements, as reported in the final results. After final tuning of the distance between the optic fiber output and the device surface, the oscilloscope tracks for the devices were acquired and stored. The oscilloscope sampling rate was set to 10 GSample/s, which was high enough for good-quality acquisition of the single 2 ns peak events. A single 200 s track therefore includes 2 × 106 data points. 3. Results and discussion Fig. 3 depicts two 200 s tracks, acquired on the reference sample (no fluorescent molecules on the SiPM surface) and on the 2 × 1013 beads/mL spotted sample. In Fig. 3a, two main differences are evident with respect to the tracks in Fig. 2, where no laser pulse was applied: • The presence of a saturation peak at the beginning of the tracks. This peak is due to simultaneous photon detection of all the 100 SiPM cells because of the very high number (about 3 × 108 ) of the incoming photons during the 2 ns laser pulse. • A much higher number of dark events following the strong initial peak, where no light is more frequent. These secondary counts (artifacts due to the afterpulsing phenomena [27]) therefore represent the real background against which the signals have to be compared. By comparing the two waveforms, the much higher signal (crowded and piled-up detection events) in the bead containing sample is clearly evident in comparison with the reference sample. The quantitative information was accessed by acquiring 21 tracks for each sample and implementing a data elaboration protocol. A suitable algorithm was developed for the offline data processing, including waveform processing consisting of detecting
L. Renna et al. / Sensors and Actuators B 209 (2015) 1011–1014
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The limit of detection can be determined via extrapolation of the concentration, generating a net signal 3 times higher than the mean standard deviation of the reference sample [28,29]. The resulting LOD is of 3.8 × 105 fluorescent nanobeads. In terms of sensitivity for a generic immunoassay, we can consider this quantity of fluorescent beads to have bonded to the active sensor surface as a consequence of the last incubation step of a microarray immunoassay. Referring to a generic immunoassay, one bead relates to the recognition of one protein on the surface. Depending on the specific biomarker molecular weight (e.g. in the 10–100 kD range) and considering the analysis volume of about 10 L usually involved in microarray applications, then the beads detection limit can be converted into a concentration LoD in a range of 1–10 pg/mL. Such sensitivity is high enough to detect important biomarkers like those related to Alzheimer disease [6], allergy detection [8] and thyroid monitoring [7].
4. Conclusions and outlook
Fig. 3. Acquired raw waveform on 10 × 10 cells, SiPM devices, subjected to 2 ns laser pulse excitation, with: (a) no nanobeads dispensed (reference sample) and (b) with nanobeads dispensed (16 drops at 2 × 1013 beads/mL). The starting laser pulse is evidenced by the intense saturated SiPM signal.
each peak against the background and subsequently integrating and summing them over a specific measurement period. Integration operations commence after the first 2 s to remove the bias of the strong after-pulsing in the first part of the curves, where the SiPM signal remains too close to the saturation value. Data analysis details are reported in supplementary information. Fig. 4 shows the results of data processing, where the net signal values (averaged signal minus averaged signal of the reference sample) and mean standard deviation over the 21 tracks are plotted. The inset shows a magnified area of the plot close to the zero signal. A reasonably dynamic range is evidenced in the scanned concentrations, although some deviation from the linearity is observed.
We have developed a novel method for biodiagnostic applications: using the active region of silicon photomultipliers as sensing areas bestows devices with several characteristics that are highly suited to the requirements of point-of-care systems, including the high integration level, low operating voltage, quantitative information with high sensitivity and good dynamic range. The system is considered compatible with the high throughput microarray technology. Microarrays of differing primary probes can be hosted on a corresponding array of micrometric SiPM detectors. Typical Integrated Circuit technology and chemical functionalization steps can be implemented to control the outermost layer chemistry to obtain the desired properties, such as binding capacity and reduction of artifact signals due to nonspecific interactions [30]. After functionalization, the SiPM surface can be precisely spotted with primary antibodies by means of conventional robotized equipment. The proposed approach is however compatible with different dispensing techniques of possible future implementation [31]. Although an oscilloscope and laser equipment was used for the above characterization, the long luminescence relaxation time and the mV range SiPM readout peaks will allow for future miniaturized apparatus developments based on LED excitation and simple reading electronics. The structure will also allow microfluidics to be easily implemented to obtain fast reactions and low reagent consumption. Finally, the device structure can be based on inexpensive elements since no expensive or bulky optical components (e.g. filters) are required for the fluorescence reading. Most of the construction steps can be implemented with the widest parallel production technology: Integrated Circuits. In the near future, we shall develop SiPM devices with smaller cells. Subsequent experiments can therefore be performed on devices smaller than 150 m × 150 m, which matches the characteristics of typical microarray spot dimensions more closely. Reduction of afterpulsing background is also being researched to obtain still higher sensitivity. Work for the implementation of immunoassay for the detection of Alzheimer’s disease using SiPMAn is currently being researched in our laboratory.
Acknowledgements Fig. 4. Plot of processed waveforms results to obtain integral photocurrent value for concentrations of nanobead solutions varying across 3 orders of magnitude and for the reference sample. The inset on the right shows a detail close to the reference signal to extract the LOD.
We are grateful to R&D Pilot Line of STMicroelectronics for manufacturing the silicon photomultiplier devices. The Italian MiUR program FIRB-MERIT is acknowledged as the founder.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2014.12.014.
[29] V. Thomsen, D. Schatzlein, D. Mercuro, Spectroscopy 18 (12) (2003) 112. [30] M. Salim, S.L. McArthur, S. Vaidyanathan, P.C. Wright, Mol. BioSyst. 7 (2011) 101. [31] C. Zheng, J. Wang, Y. Pang, J. Wang, W. Li, Z. Ge, Y. Huang, Lab Chip 12 (2012) 2487.
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Lucio Renna received the M.S. degree in Chemistry and the Ph.D in Material Science from University of Catania, in 1999 and 2007 respectively. Since 1999 he joined STMicroelectronics, where he currently works in R&D department. During his experience in STMicroelectronics, he gained multidisciplinary competencies in the development of integrated devices for bio-diagnostic applications and since 2011 is member of technical staff at STMicroelectronics. His research activity is documented by more than 40 publications and patents. Clelia Galati graduated in chemistry with honors at the University of Catania in 1999. In the same year she received a fellowship for research activity at STMicroelectronics. In 2000, he joined STMicroelectronics, where she currently works in R&D department. She performed activity as researcher in the field of molecular electronics and chemical surface functionalization. Her current activity is mainly focused on development of biosensors, in health and environment field, and as supervisor for Research Unit STMicroelectronics in the frame of national projects. Her research activity is documented by more than 40 scientific papers and patents. Natalia Spinella graduated in Chemistry with honors at the University of Catania in 2000. During the first year of her degree she started an ongoing collaboration with the University of Catania, working with the chemical physic research group. Here, she has performed research activities focused on the surface characterization of polymeric, biological and inorganic materials by time-of-flight secondary ion mass spectrometry analysis. In 2004, she joined STMicroelectronics, where she currently works as researcher in the field of chemical and biological sensor. Her research activity is documented by more than 10 publications and patents. Massimo Mazzillo received the M.S. degree with honors in physics from the University of Bari, in 2002. Since 2002, he has been with R&D STMicroelectronics, Catania, as optical sensors designer. Most of his research activity has been focused on the development of silicon single-photon detectors for biomedical applications and high-energy physics experiments and silicon carbide photodiodes for ultraviolet light detection. He is author of 14 patents and more than 80 papers published in international journals and conference proceedings. He serves as a Referee for the leading journals in the field of solid-state optical detectors and is member of STMicroelectronics Technical Staff. Salvatore Abbisso received the M.S. degree in electronics engineering from the University of Catania, Italy, in 2000. Since 2000, he has been with R&D, IMS, STMicroelectronics, Catania. Piero Giorgio Fallica is the Manager of “Advanced Sensors Development Group” inside R&D department of STMicroelectronics in Catania (Italy). He has received the degree in Physics from the University of Catania, Italy, in 1978. In 1981 he joined STMicroelectronics. He has more than 30 years’ experience in ICs, power electronics and sensors design. Starting from 1988, he managed the development of silicon sensors, mainly nuclear particle detectors and photo-sensors. International Patents issued: 11. Scientific papers and contributions to conferences: more than one hundred.