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Silicon photomultiplier (SPM) detection of low-level bioluminescence for the development of deployable whole-cell biosensors: Possibilities and limitations
Abstract
Whole-cell bacterial bioreporters await miniaturized photon counting modules with high sensitivity and robust compatible hardware to fulfill their promise of versatile, on-site biosensor functionality. In this study, we explore the photon counting readout properties of the silicon photomultiplier (SPM) with a thermoelectric cooler and the possibilities of detecting low-level bioluminescent signals. Detection performance was evaluated through a simulated LED light source and the bioluminescence produced by the genetically engineered Pseudomonas fluorescens bacterial bioreporter 5RL. Compared with the conventional photomultiplier tube (PMT), the results revealed that the cooled SPM exhibits a wider linear response to inducible substrate concentrations (salicylate) ranging from 250 to 5000 ppb. Although cooling of the SPM lowered dark count rates and improved the minimum detectable signal, and the application of a digital filter enhanced the signal-to-noise ratio, the detection of very low light signals is still limited and remains a challenge in the design of compact photon counting systems.
... Efficiency of automatic analysis procedure __________________________________ -90 -3. 4 ...
... tens of ps single photon timing resolution) and insensitivity to magnetic field (i.e. confirmed up to 7 T), the SiPM is used today in many applications like high energy physics [1] and neutrino experiments [2], fluorescence detection [3], and biophotonics [4] and medical applications [5] [6]. ...
... (2) is connected to the probe so that the signal is transmitted to a photo-detection module (3). The resulting electric signal is amplified, threshold, and integrated in the interface module (4) and then transmitted to an acquisition board on a portable PC (5). The signal is finally processed and displayed using a LabView software interface providing online measurement of the radioactivity concentration evolution in the detection volumes surrounding each implanted probe. ...
Silicon PhotoMultiplier (SiPM) detector has become a suitable visible light/photon detector for many applications like high energy physics and neutrino experiments, fluorescence detection, bio-photonics and medical imaging. The first part of my thesis was oriented to the studies of SiPM physics parameters as a function of temperature. Particularly, recent KETEK devices (year 2015) with different technological characteristics like p/n and n/p junctions, with and without trench technology, and different widths of epitaxial layer were studied in the temperature range from 308.15 K (+35°C) down to 238.15 K (-35°C). In addition, the Hamamatsu devices from 2011 production run as well as new devices from 2015 year, with improved technological characteristics inducing a reduced noise, were investigated in a wider temperature range from 318.15 K (+45°C) down to 98.15 K (-175°C). For these purposes, I participated to the design, installation, commissioning and calibration of a cryogenic experimental setup dedicated to electrical, optical and temperature studies of SiPM devices. Also, I have developed an automatic analysis procedure able to handle in a short time an impressive quantity of experimental data (i.e. tens of Gb/device) and to give a precise and fast information on main SiPM parameters and their temperature dependence. I have also developed a physical modeldescribing the DC I-V curves of SiPM detectors at different temperatures. The proposed model fits well the shape of IV curve in a very large currents range from 10⁻¹² A up to 10⁻⁵ A over the full working range of various devices. Consequently, the IV model can be used as a simple and fast method for determination of SiPM parameters like breakdown voltage VBD, the shape of Geiger triggering probability PGeiger as a function of Vbias as well as the Vbias working range. The comparison of these parameters with those calculated from AC measurements and analyzed by the automatic procedure showed a good agreement. The second part of my thesis was oriented to the study of SiPM devices and their physical parameters required to build a prototype of betasensitive intracerebral probe. Such probe is dedicated to measure the local concentration of radiolabeled molecules on awake and freely moving animal and to study new animal models of human disorders (neurodegenerative diseases, tumor growth, and neuropsychiatric disorders). It is composed of small size, low-noise SiPM device coupled to a scintillating fiber and readout by a dedicated miniaturized low-power consumption electronics. Three SiPM devices have been chosen as the most adapted for our application: two small KETEK devices of 0.5×0.5 mm² size (with and without optical trenches, specially developed by KETEK to fulfill our requirements) and a standard Hamamatsu device of 1.3×1.3 mm² size, all devices having 50 × 50 μm² μcell size. For each SiPM the gain G, dark count rate DCR and beta sensitivity were measured as a function of Vbias and temperature. The obtained results showed that the small field of view and newly developed structure of the KETEK devices allow a large decrease of the dark count rate DCR. However, this small field of view also leads to a reduced light collection due to the thickness of the epoxy protection resin on top of the SiPM and the acceptance angle of the fiber. Since the beta sensitivity represents a tradeoff between photon detection efficiency PDE and dark count rate DCR, KETEK SiPMs exhibit similar performances in comparison with the Hamamatsu device. Preliminary results demonstrate that the beta sensitivity of KETEK devices can be significantly improved by using focusing lens between the scintillating fiber and the SiPM or by reducing the thickness of its epoxy protection resin.
... One of the first comparisons of PMT vs SiPM for BL detection was performed by Li et al. who reported that PMT provided the lowest limit of detection (LOD), while cooled SiPM exhibited a wider linear range using genetically engineered BL Pseudomonas fluorescens bioreporters [63]. After a preliminary investigation of photoncounting performance using a simulated light emitting diode (LED) source, they measured BL emitted by a bacterial strain (Pseudomonas fluorescens harboring a plasmid with luxCDABE gene cassette from Aliivibrio fischeri) with a PMT with a photosensitive area of 0.5 cm 2 (H7467 series Hamamatsu) and a SiPM consisting of an array of 400 APDs each with cell size of 50 Â 50 mm and photosensitive area of 0.01 cm 2 . ...
Bioluminescence, that is the emission of light in living organisms, has been extensively explored and applied for diverse bioanalytical applications, spanning from molecular imaging to biosensing. The unprecedented technological evolution of portable light detectors opened new possibilities to implement bioluminescence detection into miniaturized devices. We are witnessing a number of applications, including DNA sequencing, reporter gene assays, DNA amplification for point-of care and point-of need analyses relying on BL. Several photon detectors are currently available for measuring low light emission, such as photomultiplier tubes (PMT), charge-coupled devices (CCD), complementary metal oxide semiconductors (CMOS), single photon avalanche diodes (SPADs), silicon photomultipliers (SiPMs) and smartphone-integrated CMOS. Each technology has pros and cons and several issues, such as temperature dependence of the instrumental specific noise, the power supply, imaging capability and ease of integration, should be considered in the selection of the most appropriate detector for the selected BL application.
These issues will be critically discussed from the perspective of the analytical chemist together with relevant examples from the literature with the goal of helping the reader in the selection and use of the most suitable detector for the selected application and to introduce non familiar readers into this exciting field.
... The detection instrument was designed to sensitively detect luminescence signal from the captured reporter on the NT membrane in the microfluidic device using a PMT, but alternative detector technologies can be considered [40][41][42][43][44][45] . A simple mechanical shutter mechanism (Fig. S1C †) was designed to shield the PMT's detector from excess ambient light when the platform's cover was open during sample introduction. ...
Current quantification methods of Escherichia coli (E. coli) contamination in water samples involve long incubation, laboratory equipment and facilities, or complex processes that require specialized training for accurate operation and interpretation. To address these limitations, we have developed a microfluidic device and portable instrument prototypes capable of performing a rapid and highly sensitive bacteriophage-based assay to detect E. coli cells with detection limit comparable to traditional methods in a fraction of the time. The microfluidic device combines membrane filtration and selective enrichment using T7-NanoLuc-CBM, a genetically engineered bacteriophage, to identify 4.1 E. coli CFU in 100 mL of drinking water within 5.5 hours. The microfluidic device was designed and tested to process up to 100 mL of real-world drinking water samples with turbidities below 10 NTU. Prototypes of custom instrumentation, compatible with our valveless microfluidic device and capable of performing all of the assay's units of operation with minimal user intervention, demonstrated similar assay performance to that obtained on the benchtop assay. This research is the first step towards a faster, portable, and semi-automated, phage-based microfluidic platform for improved in-field water quality monitoring in low-resource settings.
... 22−24 Silicon photomultipliers (SiPMs) have also been proposed as sensitive light detectors for BL. 25,26 SiPMs are arrays of avalanche photodetectors working in Geiger mode (GM-APDs), each one having integrated passive-quenching resistor and connected in parallel. 27 Due to their high quantum efficiency, low energy and bias voltage requirements, and rapid fast response time (in nanosecond time scale), SiPMs have attracted significant interest for various applications, providing in some cases better performance than smartphones. ...
The availability of portable analytical devices for on-site monitoring and rapid detection of analytes of forensic, environmental, and clinical interest is vital. We report the development of a portable device for the detection of biochemiluminescence relying on silicon photomultiplier (SiPM) technology, called LuminoSiPM, which includes a 3D printed sample holder that can be adapted for both liquid samples and paper-based biosensing. We performed a comparison of analytical performance in terms of detectability with a benchtop luminometer, a portable cooled charge-coupled device (CCD sensor), and smartphone-integrated complementary metal oxide semiconductor (CMOS) sensors. As model systems, we used two luciferase/luciferin systems emitting at different wavelengths using purified protein solutions: the green-emitting P. pyralis mutant Ppy-GR-TS (λmax 550 nm) and the blue-emitting NanoLuc (λmax 460 nm). A limit of detection of 9 femtomoles was obtained for NanoLuc luciferase, about 2 and 3 orders of magnitude lower than that obtained with the portable CCD camera and with the smartphone, respectively. A proof-of-principle forensic application of LuminoSiPM is provided, exploiting an origami chemiluminescent paper-based sensor for acetylcholinesterase inhibitors, showing high potential for this portable low-cost device for on-site applications with adequate sensitivity for detecting low light intensities in critical fields.
... These traits have brought a lot of interest in the medical area, such as x-ray imaging, medical imaging, and neutron science [9][10][11][12][13][14]. The SiPM has numerous advantages, such as requiring a low voltage, and does not incur damage when exposed to high levels of external light [15,16]. Based on these characteristics, the development of an SiPM-based high-sensitivity luminometer for small volume samples with a low cost for luminescence-based assays shows promise [17]. ...
The silicon photomultiplier (SiPM) for low light detection has many advantages when compared to existing photon counting detectors, such as high sensitivity, low cost, robustness, and compact hardware. To facilitate the use of SiPM as a portable, field deployable device, an electrical circuit was designed consisting of an amplifier, comparator, and microcontroller. In addition, a 3D printing was used to create a portable cradle for housing the SiPM. To evaluate its detection ability, a laser experiment and bioluminescent experiments, including Pseudomonas fluorescens M3A detection, E. coli O157:H7 PhiV10nluc lysogen detection, and a luminescence-based detection of E. coli O157:H7 in ground meat using the engineered luminescent-based reporter phage PhiV10nluc, were conducted. In the same experimental setting, our previously developed smartphone-based luminometer called the bioluminescent-based analyte quantitation by smartphone and a conventional photomultiplier tube-based benchtop luminometer were used to compare detection levels and applicability for supporting luminescent phage-based pathogen detection. Results showed that the SiPM provides better performance in terms of time to detection and SNR and could be used as the light detection component of the PhiV10nluc phage-based detection format.
... SiPM potential is being currently studied for biomedical application [26], compact imaging systems [27,28], etc. by a lot of research groups in the world [29,30]. Sensitivity of the SiPM detector depends on the ambient temperature, which can be overcome by using a photodetector cooling system [31]. ...
Enzymatic luminescent systems are a promising tool for rapid detection of heavy metals ions for water quality assessment. Nevertheless, their widespread use is limited by the lack of test procedure automation and available sensitive handheld luminometers. Herein we describe integration of disposable microfluidic chips for bioluminescent enzyme-inhibition based assay with a handheld luminometer, which detection system is based on a thermally stabilized silicon photomultiplier (SiPM). Microfluidic chips were made of poly(methyl methacrylate) by micro-milling method and sealed using a solvent bonding technique. The composition of the bioluminescent system in microfluidic chip was optimized to achieve higher luminescence intensity and storage time. Results indicate that developed device provided comparable sensitivity with bench-scale PMT-based commercial luminometers. Limit of detection for copper (II) sulfate reached 2.5 mg/L for developed biosensor. Hereby we proved the concept of handheld enzymatic optical biosensors with disposable chips for bioassay. The proposed biosensor can be used as an early warning field-deployable system for rapid detection of heavy metals salts and other toxic chemicals, which affect bioluminescent signal of enzymatic reaction.
... It is expected that in the future SiPM could also have a role in replacing imaging devices in radiotherapy. Other fields where it is expected to have a high demand of SiPMs are biosensor devices [129] and point of care applications. Thanks to the small size and low cost, the SiPM could really represent the enabling technology for these fields that are rapidly emerging in the medical sector. ...
Silicon photomultipliers (SiPMs) are becoming the reference photodetectors in many fields. In medicine they are slowly replacing photomultiplier tubes and avalanche photodiodes in medical imaging and in PET in particular. In this paper a broad overview of the current applications of SiPM in medicine is presented. The major fields where the SiPMs are used, namely PET/MR and hadrontherapy are discussed at length.
... Distributed sensors preferably use Optical Time Domaine Reflectometry (OTDR) detection. The principle of OTDR is described by Marcuse in [5,[64][65][66][67]. ...
This review summarizes principles and current stage of development of fiber-optic chemical sensors (FOCS) and biosensors (FOBS). Fiber optic sensor (FOS) systems use the ability of optical fibers (OF) to guide the light in the spectral range from ultraviolet (UV) (180 nm) up to middle infrared (IR) (10 μm) and modulation of guided light by the parameters of the surrounding environment of the OF core. The introduction of OF in the sensor systems has brought advantages such as measurement in flammable and explosive environments, immunity to electrical noises, miniaturization, geometrical flexibility, measurement of small sample volumes, remote sensing in inaccessible sites or harsh environments and multi-sensing. The review comprises briefly the theory of OF elaborated for sensors, techniques of fabrications and analytical results reached with fiber-optic chemical and biological sensors.
... SensL products are designed for use in high-volume markets, such as medical imaging positron emission tomography (PET), [1][2][3][4][5] hazard and threat radiation detection, 6-8 highenergy physics, automotive driver assist systems (ADAS), using laser detection and range finding (LIDAR) and biophotonics. [9][10][11] The purpose of this paper is to review the basic SensL SiPM sensor, to outline the raw sensor test parameters, to demonstrate the performance in application-relevant environments, and to show uniformity data that have been obtained during mass production wafers runs. Additionally, it is desired to show the outcome of recent reliability testing of SensL B-Series sensors using integrated circuit industry standard test procedure. ...
This publication details CMOS foundry fabrication, reliability stress assessment, and packaged sensor test results obtained during qualification of the SensL B-Series silicon photomultiplier (SiPM). SiPM sensors with active-area dimensions of 1, 3, and 6 mm were fabricated and tested to provide a comprehensive review of SiPM performance highlighted by fast output rise times of 300 ps and photon detection efficiency of greater than 41%, combined with low afterpulsing and crosstalk. Measurements important for medical imaging positron emission tomography systems that rely on time-of-flight detectors were completed. Results with LSYO:Ce scintillation crystals of 3 × 3 × 20 mm3 demonstrated a 225 Æ 2-ps coincidence resolving time (CRT), and the fast output is shown to allow for simultaneous acquisition of CRT and energy resolution. The wafer level test results from ∼150 k 3-mm SiPM are shown to demonstrate a mean breakdown voltage value of 24.69 V with a standard deviation of 0.073 V. The SiPM output optical uniformity is shown to be +- 10% at a single supply voltage of 29.5 V. Finally, reliability stress assessment to Joint Electron Device Engineering Council (JEDEC) industry standards is detailed and shown to have been completed with all SiPM passing. This is the first qualification and reliability stress assessment program run to industry standards that has been reported on SiPM.
Point-of-care testing (POCT) technology is highly desirable for clinical diagnosis, healthcare monitoring, food safety inspection, and environment surveillance, because it enables rapid detection anywhere, anytime, and by anyone. Electrochemiluminescence (ECL) has been widely used in chemo-/bio analysis due to its advantages such as high sensitivity, simplicity, rapidity and easy to control, and is now attracting increasing attention for POCT applications. However, to realize the accurate on-site quantitation, it is still challenging to develop portable devices which can precisely collect, analyze, transmit and display the ECL signals. This review will focus on how to develop a portable ECL device by summarizing recent examples and analyzing their key components part by part. Then the possible solutions to the existing challenges in the development and applications of portable ECL devices are summarized and discussed in detail, followed by offering future perspectives. We attempted to provide an appealing viewpoint to inspire interested researchers to comprehend and explore portable ECL sensing systems for practical applications and even commercialization.
An electrochemiluminescence (ECL) analytical device was developed using an electric contactless power transfer system. A mutually induced electromotive voltage was generated by wrapping an enameled wire around a commercial contactless charger. There was no electrical contact between the power supply and the electrochemical cell. For the tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)32+)/tri-n-propylamine system, a weak ECL signal was observed. When an inexpensive rectifier diode was introduced between the coil and the working electrode, the ECL intensity detection sensitivity increased by more than 100 times. The relationship between the waveform of the applied voltage and the ECL response was clarified, and the optimum conditions were determined. The intensity of the induced electromotive voltage was easily controlled by changing the number of turns in the coil. The proposed method is a safe, simple, and inexpensive technique without electrical contact.
We developed a miniaturized electrochemiluminescence (ECL) instrument coupled with a light‐emitting diode‐based bipolar electrochemical sensor (LED‐BPES). This instrument composes of a microcontroller circuit, a power supply circuit, a potentiostat, an optical detecting circuit, and a communication circuit. The multi‐pixel photon counter (MPPC), which is low‐cost, small‐size, and wide‐range in optical measurements, is chosen as the optical detector. The LED‐BPES composes of a disposable screen‐printed carbon electrode (SPCE) and a surface‐mount red LED. Depended on the closed bipolar electrode (C‐BPE) structure, the LED‐BPES not only avoids the employment of unstable and complex ECL reactions but also offers a cost‐effective alternative for the over‐priced ECL reagents by using a mini‐size commercial LED as the luminescent producer. The combination of MPPC and LED‐BPES helps to set up the simplified and downsized instrument system with low price and high efficiency. The presented instrument coupled with LED‐BPES works excellent in electroactive molecules detection and has great potential in the application of heavy metal ions detection.
3D cell culture models represent an attractive approach to decode intracellular and intercellular signaling, providing biologically relevant information and predictive data. Bioluminescent reporter gene assays and bioluminescence imaging in 3D cell models are very promising bioanalytical tools for several applications.Here we report a very straightforward method for bioluminescence imaging and bioluminescent reporter gene assays in 3D cell-culture models. Both the assays can be easily implemented in laboratories equipped with basic cell culture facilities and instrumentation for bioluminescence detection, that is, low-light detectors connected to inverted microscopes and luminometers, without the need for additional equipment.
A readout circuit dedicated for the detection of the chemiluminescence phenomenon using a silicon photomultiplier (SiPM) is presented. During chemiluminescence, light is generated as a result of chemical reaction. Chemiluminescence is used in many applications within medicine, chemistry, biology and biotechnology, and is one of the most important sensing techniques in biomedical science and clinical medicine. The front-end electronics consist of a preamplifier and a fast shaper—this produces pulses, the peaking time which is 3.6 ns for a single photon and the FWHM is 3.8 ns. The system has been optimised to measure chemiluminescence—it is sensitive at the level of single photons, it generates a low number of overlapping pulses and is accurate. Two methods of signal detection are analysed and compared: the counting of events and amplitude detection. The relationship between the chemiluminescence light intensity and the concentration of the chemical compound (luminol) is linear in the range of the tested concentrations and has strong linearity parameters and low prediction intervals.
SensL C-Series Silicon Photomultiplier (SiPM) sensors are fabricated in a high-volume CMOS foundry to a custom SensL process, and packaged as a reflow solderable surface mount device. Advances in SiPM production have resulted in significant improvement in PDE, dark current as well as tighter breakdown voltage uniformity for the C-Series SiPM sensors. The SiPM are fabricated with a shallow P-on-N junction optimized for the detection of shorter wavelength photons, with a peak PDE of 41% at 420nm and excellent sensitivity extending to wavelengths <300nm. The dark currents have been reduced through the reduction of damage during semiconductor processing and an order of magnitude reduction has been achieved. The breakdown voltage variation has been improved through process optimization to minimize variations. With these process improvements typical dark count rates of ∼30kHz/mm2 are achieved simultaneously with breakdown voltage uniformity of ±213mV demonstrated. In addition, application specific measurements of CRT (Coincidence Resolving Time) that are relevant to PET (positron emission tomography) will be shown to be 210ps at 7.5V overvoltage. In addition to device characterization work, this paper will address the wafer-level fabrication and testing, package level testing required by high volume SiPM sensor applications.
Bioluminescence is light production by living organisms, which can be observed in numerous marine creatures and some terrestrial invertebrates. More specifically, bacterial bioluminescence is the “cold light” produced and emitted by bacterial cells, including both wild-type luminescent and genetically engineered bacteria. Because of the lively interplay of synthetic biology, microbiology, toxicology, and biophysics, different configurations of whole-cell biosensors based on bacterial bioluminescence have been designed and are widely used in different fields, such as ecotoxicology, food toxicity, and environmental pollution. This chapter first discusses the background of the bioluminescence phenomenon in terms of optical spectrum. Platforms for bacterial bioluminescence detection using various techniques are then introduced, such as a photomultiplier tube, charge-coupled device (CCD) camera, micro-electro-mechanical systems (MEMS), and complementary metal-oxide-semiconductor (CMOS) based integrated circuit. Furthermore, some typical biochemical methods to optimize the analytical performances of bacterial bioluminescent biosensors/assays are reviewed, followed by a presentation of author’s recent work concerning the improved sensitivity of a bioluminescent assay for pesticides. Finally, bacterial bioluminescence as implemented in eukaryotic cells, bioluminescent imaging, and cancer cell therapies is discussed.
Graphical Abstract
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.
The need for decentralized clinical tests together with the concept of time and cost saving are pushing the development of portable, miniaturized, compact biosensors with diagnostic and prognostic purpose. Here, we propose an innovative detection system based on a Single Photon Avalanche Diode (SPAD) with high sensitivity and low noise, crucial features for an efficient chemiluminescence biosensor. The SPAD detector, having 60µm diameter, has a Photon Detection Efficiency higher than 55% at 425nm and a Dark Count Rate lower than 100Hz at room temperature. Our design allows a good optical coupling efficiency between sample and detector. A specific biofunctional surface was implemented taking advantage of aptamers, short DNA sequences having high selectivity and affinity toward their targets. We successfully detected physiological levels of Vascular Endothelial Growth Factor (VEGF), a circulating protein biomarker highly correlated with cancer. The SPAD aptasensor showed a Limit of Detection (LoD) in the pM range, stability (up to 42 days) and re-usability (up to seven cycles). This compact biosensor is therefore a promising step toward the actual use of portable microdevices in diagnostics.
Copyright © 2015 Elsevier B.V. All rights reserved.
The use of smart supports and bioinspired materials to confine living cells and use them for field-deployable biosensors has recently attracted much attention. In particular, bioluminescent whole-cell biosensors designed to respond to different analytes or classes of analyte have been successfully implemented in portable and cost-effective analytical devices. Significant advances in detection technology, biomaterial science, and genetic engineering of cells have recently been reported. Now the challenge is to move from benchtop traditional cell-based assays to portable biosensing devices. Improvement of the analytical performance of these biosensors depends on the availability of optimized bioluminescent reporters, and promising approaches that go beyond reporter gene technology are emerging. To enable handling of cells as ready-to-use reagents, nature-inspired strategies have been used, with the objective of keeping cells in a dormant state until use. Several issues must still be investigated, for example long-term viability of cells, the possibility of performing real-time analysis, and multiplexing capability.
Figure
Concept of whole-cell bioluminescent biosensor
Geiger-mode avalanche photodiodes (APD) or photon counting detectors (PCD) have become the basis of a range of new detectors and applications. Such devices are sensitive to single photons and are being adapted to create different detector technologies such as the silicon photomultiplier (SPM). A silicon photomultiplier, so-called because of its similarity in performance to conventional photomultiplier tubes, is based on an array of photon counting detectors but with a single common output. While the silicon photomultiplier does not provide positional information, it does allow photon number resolution and photon counting at higher count rates than are achievable with a solitary PCD. These new detectors are enabling a range of new applications in the fields of medicine, biology, high-energy physics and space exploration. In this paper we report the performance of PCD with dimensions ranging from 20 μm × 20 μm to 200 μm × 200 μm and on 1 mm2 silicon photomultipliers and illustrate the use of an SPM in single photon counting mode.
Whole-cell, genetically modified bioreporters are designed to emit detectable signals in response to a target analyte or related group of analytes. When integrated with a transducer capable of measuring those signals, a biosensor results that acts as a self-contained analytical system useful in basic and applied environmental, medical, pharmacological, and agricultural sciences. Historically, these devices have focused on signaling proteins such as green fluorescent protein, aequorin, firefly luciferase, and/or bacterial luciferase. The biochemistry and genetic development of these sensor systems as well as the advantages, challenges, and common applications of each one will be discussed.
We report a compact biosensor using genetically engineered whole-cell bioreporters on a CMOS based integrated circuit, also called the microluminometer, for accurately sensing low concentrations of a wide range of toxic substances in both gas and liquid environments. The bioluminescent bioreporters are bacteria that can be genetically altered to achieve bioluminescence when in contact with a targeted substance. The bioreporters are placed in close proximity to the microluminometer thus obviating the need for complex instrumentation to channel light from the bioreporters to the microluminometer. The microluminometer presented here is a more robust design and consumes lower power when compared to the one presented in Bolton et al.'s work [E.K. Bolton, G.S. Sayler, D.E. Nivens, J.M. Rochelle, S. Ripp, M.L. Simpson, Integrated CMOS photodetectors and signal processing for very low-level chemical sensing with the bioluminescent bioreporter integrated circuit, Sens. Actuators B 85 (2002) 179–185]. The microluminometer includes integrated photodiodes and signal processor and is realized using a standard 0.35 μm CMOS process. The photodiode array is operated with a reverse bias of a few mV and demonstrates a leakage current of 45–60 fA and a minimum detectable signal (MDS) of 0.15 fA while operating at room temperature. We present results from both liquid based and air based testing with salicylate and naphthalene as the analytes, respectively, and genetically altered bacteria, Pseudomonas fluorescens 5RL, as the bioreporter.
The bioluminescently marked Pseudomonas fluorescens strain 5RL, has been used previously to follow colonisation of soy bean roots (De Weger et al. [1991] Appl. Environ. Microbiol. 57:36-41). In the present paper the method has been further developed and optimized for wheat roots and it is used to get a quick overview of the colonisation patterns of many different root systems at the same time. Colonisation was followed on wheat plants grown in our gnotobiotic sand system (Simons et al., 1996. Mol Plant Microbe Interact 9: 600-607) and the following results were obtained. (i) A spatio-temporal analysis of the colonisation of wheat roots showed that 4 days after planting the highest bacterial activity was observed at the upper part of the root. After 6 days the high bacterial activity at the upper part was further increased, whereas spot-like activities were observed on the lower root parts, possibly due to micro-colonies. (ii) Bacterial mutations causing lack of motility or auxotrophy for amino acids resulted in impaired colonisation of the lower root parts, indicating that motility and prototrophy for the involved amino acid(s) are important factors for wheat root colonisation by strain 5RL. (iii) Coinoculation of strain 5RL with other wild type Pseudomonas strains on the root influenced the colonisation pattern observed for strain 5RL. Colonisation was not visually affected when the competing strain was a poor root coloniser, but was severely reduced when the competing strain was a good root coloniser. The results show that the spatio-temporal colonisation of wheat root by P. fluorescens strain 5RL and derivatives is similar to that of strain WCS365 on tomato. The advantage of the use of lux-marked strains is that the results are obtained much quicker than when conventional methods are used and that the result is supplied as an image of the colonisation pattern of many different roots.
This study concerns the formation of structured communities by pure cultures and binary associations of Pseudomonasfluorescens transgenic strains and natural heterotrophic bacterial species in naphthalene-containing media with various osmotic pressures. It was shown that cells of P. fluorescens strain 5RL, harboring a recombinant construct in the chromosome, were more resistant to the combined action of the stress factors under study than P. fluorescens 82/pUTK21, harboring a recombinant construct within a plasmid. Natural P. fluorescens 1 strains, particularly Vibrio sp. 14, were more viable at high osmotic pressures and naphthalene concentrations. Experiments with the combined introduction of transgenic and natural bacterial strains at high osmotic pressures demonstrated the stable coexistence of bacterial associations in biofilms, independent of naphthalene concentration. Strains considered for introduction into the environment for bioremediation should be assessed with regard to their susceptibility to the combined effect of anthropogenic and natural stress factors. The design of bacterial associations for the same purpose should take into account the effect of factors important for their survival in polluted areas.
Silicon Photo-Multiplier (SiPM) is a novel type of avalanche photon detector which operates in Geiger mode. It allows great advancement in photon detection and is a good candidate for replacing traditional photomultiplier tubes (PMTs). In this paper the current status of development of a research project founded by the Italian National Institute for Nuclear Physics (INFN) will be presented, in the framework of the DASIPM collaboration which involves several Universities and the Center for Scientific and Technological Research (FBKirst, Trento, Italy). Recent advancements in SIPM technology will be presented, along with some preliminary results and a discussion about possible exploitations in medical and astroparticle physics applications.
A novel detector called silicon multiplier has been developed that replaces the photomultiplier tube in many applications. The detector uses a large parallel array of silicon photon-counting diodes and integrated quenching elements to provide single-photon analog mode sensitivity for low-light sensing. Incident photons are detected by the single-photon-counting diodes in the array, which avalanche and release a fixed quantity of charge onto the output node. The quenching element which is the resistor quenches the avalanche and resets the diode. The output detector therefore is made up of the charge contributions from all single-photon-counting diodes in the array. These detectors operate on low voltages of less than 100 V, have high quantum efficiency, immune to magnetic fields, have uniform gain across the array, robust with no damage from high light levels and are small in size.
An Escherichia coli strain, genetically modified to emit a luminescent signal in the presence of genotoxic agents, was alginate-immobilized onto an exposed core of a fiber-optic. The performance of this whole-cell optical fiber sensor system was examined as a function of several parameters, including gel probe matrix volume, bacterial cell density, numerical aperture of the fiber core and working temperature. An optimal response to a model genotoxicant, mitomycin C, was achieved with six alginate/bacterial adlayers on a 1 cm exposed fiber-optic core. Total alginate volume per tip was about 100 ml, containing a bacterial suspension of around 1.5–3:0 Â 10 7 cells. When the core diameter was etched down to 270 mm, photon detection efficiency significantly increased, although to a lesser extent than that expected from theoretical calculations. Further reduction in core diameter led to a reduced performance. Activity at 378C was superior to that at 268C. Under these optimized conditions, optrode response was mitomycin C dose-dependent for at least 6 h, with a lower detection threshold of 25 mg/l. # 2001 Elsevier Science B.V. All rights reserved.
Promoters for stress response genes have been fused to the luxCDABE cassette to produce bioreporters that respond to a number of different types of environmental and chemical stresses (Table 1). These types of bioreporters are not used to identify a particular stressor, but simply that the stress response has been activated. For example, heat shock promoters in bioreporters have been used to respond not just to conditions involving a change in temperature, but also to detect exposure to chemical and biochemical stressors such as antibiotics, organic compounds, oxidative reagents and heavy metals (Kregel 2002). For example, Belkin et al. (1997) constructed five plasmids containing different promoter–luxCDABE fusions and used the plasmids to transform an E. coli strain into a panel of bioluminescent bioreporters for monitoring stress responses. Promoters for katG and soxRS for oxidative stresses, recA for DNA damage, grpE for the heat shock/protein damage system and fabA involved in fatty-acid synthesis inhibition were used. The bioreporters reacted specifically to stressors within their respective groups, except for phenol, which nonspecifically induced all of the strains. Davidov et al. (2000) offered potential improvements in the SOS-type recA promoter-luxCDABE bioreporter response by constructing strains that (i) deactivated the expression of an efflux pump (tolC mutation) to improve sensitivity, (ii) provided a more stable construct by incorporating the promoter–lux fusion into the chromosome and (iii) used the luxCDABE genes from P. luminescens, allowing use at higher temperatures. A plasmid containing an SOS dependent promoter–luxCDABFE fusion (the lux genes were isolated from Photobacterium leiognathi) has been introduced into the Salmonella typhimurium TA1535 that was used in the Ames test for mutagenesis (Rabbow et al. 2002). The system gave comparable results to the MutatoxTM (Strategic Diagnostics Inc., Newark, NJ, USA), Ames and SOS-chemotests assays.
Reporter strains of bacteria were tested using soil samples from several sites near a leaking fuel oil storage facility. The
reporter bacteria utilized the bioluminescentlux genes fromVibrio fischeri, which were transcriptionally fused to catabolic gene sequences. The catabolic genes of interest specified the degradation
of toluene (from the TOL plasmid) and naphthalene (from the NAH7 plasmid and from a NAH plasmid recently isolated). The results
indicated that two soil samples were contaminated with both toluene (or xylene) and naphthalene. These data were useful in
describing the extent of contamination at the site.
A robust neutron detector was developed to monitor the neutron flux in fast-neutron/gamma radiography (FNGR) cargo scanners. The main features of the new neutron detector were an ability to handle high count rate for statistical precision and a high damage threshold for longevity when used close to intense fast neutron sources (up to 10<sup>10</sup> n/s). The detector comprises a fast scintillator coupled to a shielded 100 pixel Hamamatsu 1 mm times 1 mm silicon photomultiplier (SiPM) via a 220 cm length of wavelength shifting (WLS) plastic fibre. The WLS fibre was glued onto two long sides of a 10 times 10 times 50 mm scintillator wrapped in Tyvek reflector. Scintillation pulses of up to ~40 photons reached the SiPM. With the detector mounted 60 mm from a Thermo A-325 DT neutron generator (up to 6 times 10<sup>7</sup> n/s) the count rate was over 4 times 10<sup>4</sup> counts per second. The SiPM gain was effectively stabilised against temperature variations by automatically trimming the bias voltage with SiPM temperature. Comparison data is provided for a photodiode-scintillator neutron detector placed 134 cm from the A-325 neutron generator and for a PMT-WLS-scintillator neutron detector.
Progress in molecular biology has made available new bioanalytical tools that take advantage of the great detectability and the simple analytical format of bioluminescence. Combining luminescent enzymes or photoproteins with biospecific recognition elements at the genetic level has led to the development of ultrasensitive, selective bioanalytical tools (e.g., recombinant whole-cell biosensors, immunoassays and nucleic-acid hybridization assays). Optical in vivo imaging is also growing rapidly, propelled by the benefits of bioluminescent tomography and imaging systems, and making inroads into monitoring biological processes with clinical, diagnostic and drug-discovery applications. Bioluminescence-detection techniques are also appropriate for miniaturized bioanalytical devices (e.g., microarrays, microfluidic devices and high-density-well microtiter plates) for the high-throughput screening of genes and proteins in small sample volumes.
Positron Emission Tomography (PET) for small animal studies requires high-resolution gamma cameras with high sensitivity. Traditionally, inorganic scintillators are used and, in recent times, coupled to position sensitive PMTs to achieve a higher resolution. Such PSPMTs are costly, operated at high voltage and have a relatively low packing fraction. However, their advantage, compared to current solid state photodetectors, is their high signal-to-noise ratio. The Silicon Photomultiplier (SiPM) is a silicon diode detector that shows great promise as a photodetector for scintillators and hence application in nuclear medicine imaging applications. The microcell MRS (Metal–Resistor–Semiconductor) structure of the SiPM leads to a self-quenching, Geiger-mode avalanche photodiode (GAPD), that produces a large gain (5×105) at low bias voltage (50 V) and proportional output for moderate photon flux. Such a compact silicon detector, with a performance similar to a PMT, is obviously well disposed to being developed into a close-packed array in order to have a position-sensitive detection surface. We propose a miniature, high-resolution camera for a small-animal PET imaging system that is based on such an array of SiPM. The design is based upon the classic Anger camera principle; each detector module consists of a continuous slab of scintillator, viewed by a matrix of SiPM. A detector head of in area is proposed, constructed from three such modules of the continuous camera described above. The stacked layers would give the system intrinsic depth of interaction (DOI) information. A summary of measured SiPM performance and results of a simulation of the proposed camera, using the Monte Carlo package GEANT4, are presented. It is shown that using three layers of 5 mm thick LSO, gives an efficiency of 68% with maximum count rates in the front layers. Intrinsic spatial resolution of FWHM was found although this is degraded at the edges. Although the inclusion of DOI information increases the overall spatial resolution, the parallax error was still found to be the limiting factor in a small animal system.
The state of art of the Silicon Photomultipliers (SiPM's)—their features, possibilities and applications—is given. The significant progress of this novel technique of photo detection is described and discussed.
The Silicon Photomultiplier (SiPM) is a semiconductor device consisting of many photon microcounters (103 mm−2) positioned on a common Si substrate. SiPM operates in a limited Geiger mode and has single photoelectron gain (106) and photon detection efficiency (20%) similar to vacuum PMT. Main SiPM features are described and a number of examples of its possible applications are demonstrated, such as scintillator fiber readout, scintillator tiles+WLS readout, imaging Cherenkov counter timing. These SiPM applications are based on experimental test data and SiPM performance is compared with other photodetectors (PMT, APD, HPD, VLPC).
We have developed a genetically encoded bioluminescent indicator for the Na(+) channel, in which Na(+)-sensitive Gaussia luciferase is fused with the voltage-gated Na(+) channel. This indicator is capable of detecting Na(+) flow through the pores of Na(+) channels. Because of high sensitivity and low background in luminescence assays, the absence of toxicity, and a wide linear dynamic range, this luciferase can be used to generate a novel, genetically encoded Na(+) channel indicator. It may provide a high-throughput screening system for drug discovery against Na(+) channels, which should be useful in controlling lethal cardiac arrhythmias, epileptic seizures, and intolerable pain associated with terminal stages of cancer. It may also offer a system for monitoring the Na(+) channel activity in living cells, which may be useful in illuminating neuronal activity in vivo.
Whole-cell biosensors are potential candidates for on-line and in situ environmental monitoring. In this work we present a new design of a whole-cell bioluminescence biosensor for water toxicity detection, based on genetically engineered Escherichia coli bacteria, carrying a recA::luxCDABE promoter-reporter fusion. Sensitive optical detection is achieved using a single photon avalanche photodiode (SPAD) working in the Geiger mode. The present work describes a simple mathematical model for the kinetic process of the bioluminescence based SOS toxin response of E. coli bacteria. We find that initially the bioluminescence signal depends on the time square and we show that the spectral intensity of the bioluminescence signal is inverse proportional to the frequency. We get excellent agreement between the theoretical model and the measured light signal. Furthermore, we present experimental results of the bioluminescent signal measurement using a SPAD and a photomultiplier, and demonstrate improvement of the measurement by applying a matched digital filter. Low intensity bioluminescence signals were measured after the whole-cell sensors were exposed to various toxicant concentrations (5, 15 and 20ppm).
We have developed a sensitive and specific method based on surface plasmon resonance (SPR) for detection of insulin autoantibodies (IAA) in serum samples from individuals at high risk of developing type 1 diabetes (T1D). When measuring trace molecules in undiluted sera with label-free techniques like SPR, non-specific adsorption of matrix proteins to the sensor surface is often a problem, since it causes a signal that masks the analyte response. The developed method is an indirect competitive immunoassay designed to overcome these problems. Today, IAA is mainly measured in radio immunoassays (RIAs), which are time consuming and require radioactively labeled antigen. With our SPR-based immunoassay the overall assay time is reduced by a factor of >100 (4 days to 50min), while sensitivity is maintained at a level comparable to that offered by RIA.
The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.
We report an integrated CMOS microluminometer optimized for the detection of low-level bioluminescence as part of the bioluminescent bioreporter integrated circuit (BBIC). This microluminometer improves on previous devices through careful management of the sub-femtoampere currents, both signal and leakage, that flow in the front-end processing circuitry. In particular, the photodiode is operated with a reverse bias of only a few mV, requiring special attention to the reset circuitry of the current-to-frequency converter (CFC) that forms the front-end circuit. We report a sub-femtoampere leakage current and a minimum detectable signal (MDS) of 0.15 fA (1510 s integration time) using a room temperature 1.47 mm2 CMOS photodiode. This microluminometer can detect luminescence from as few as 5000 fully induced Pseudomonas fluorescens 5RL bacterial cells.
The detection of microorganisms in root canals is generally limited to qualitative or semiquantitative methods. We describe a new and nondestructive in vitro method to quantify root-canal bacteria over sequential treatment procedures using real-time imaging in conjunction with the bioluminescent reporter strain Pseudomonas fluorescens 5RL. Induced bacterial photon emission can be monitored by sensitive optical photonic imaging and luminometry, providing images as well as spatial and quantitative measurements. Bioluminescence imaging and luminometry determined that the lower limit of detection of bacteria in root canals occurred between 2 x 10(2) and 2 x 10(3) cells, with high correlation between cell counts and detection devices (r > or = 0.981). A preliminary study assessed the efficacy of sequential irrigation procedures to remove 5 x 10(6) bacteria from root canals (n = 5; apical size 60) using a 28-gauge, endodontic needle positioned 1 mm from working length; 9.2% +/- 3.1% and 8% +/- 3.6% of bacteria remained after 3 and 6 ml irrigation, respectively (p = 0.03), corresponding to approximately 4 x 10(5) bacteria remaining after 6 ml. This method can be used to study the efficacy of sequential endodontic treatment procedures in removing bacteria from root canals.
To test the hypothesis that the mechanical efficacy of irrigation in reducing bacteria in the root canal is dependent on depth of placement of the irrigation needle.
The root canals of 30 permanent cuspids were instrumented to apical size 60 using a crown-down technique. A suspension of the bioluminescent reporter strain Pseudomonas fluorescens 5RL was inoculated into each canal of sterilized teeth. Emission of bioluminescence (photons s(-1)) from each tooth was quantified on four sequential occasions using luminometry and bioluminescence imaging: (i) background, (ii) after inoculation, (iii) after irrigating the inoculated teeth with 3 mL of a nonantimicrobial irrigant delivered either 1 mm (group 1, n = 15) or 5 mm (group 2, n = 15) from working length (WL) using a 28G safety-ended irrigating needle, (iv) after an additional 3 mL irrigation (total 6 mL). Intragroup and intergroup comparisons were made using Wilcoxon matched pairs and Mann-Whitney tests, respectively.
In group 1, there was a mean log10 decrease in bacteria of 0.68 +/- 0.26 after 3 mL of irrigant compared with 1.19 +/- 0.48 after 6 mL (P < 0.001); in group 2 the mean log10 decrease was 0.58 +/- 0.28 after 3 mL of irrigant compared with 0.69 +/- 0.35 after 6 mL (P < 0.02) (Wilcoxon matched pairs). Using 3 mL of irrigant, needle depth did not have a significant effect on reduction of intracanal bacteria (P = 0.407), but the effect became significant when 6 mL of irrigant was used (P < 0.002) (Mann-Whitney tests).
The mechanical efficacy of 6 mL of irrigant in reducing intracanal bacteria was significantly greater when delivered 1 mm compared with 5 mm from WL.
Microorganisms will be an integral part of biologically based waste processing systems used for water purification or nutrient recycling on long-term space missions planned by the National Aeronautics and Space Administration. In this study, the function and stability of microbial inocula of different diversities were evaluated after inoculation into plant-based waste processing systems. The microbial inocula were from a constructed community of plant rhizosphere-associated bacteria and a complexity gradient of communities derived from industrial wastewater treatment plant-activated sludge. Community stability and community function were defined as the ability of the community to resist invasion by a competitor (Pseudomonas fluorescens 5RL) and the ability to degrade surfactant, respectively. Carbon source utilization was evaluated by measuring surfactant degradation and through Biolog and BD oxygen biosensor community level physiological profiling. Community profiles were obtained from a 16S-23S rDNA intergenic spacer region array. A wastewater treatment plant-derived community with the greatest species richness was the least susceptible to invasion and was able to degrade surfactant to a greater extent than the other complexity gradient communities. All communities resisted invasion by a competitor to a greater extent than the plant rhizosphere isolate constructed community. However, the constructed community degraded surfactant to a greater extent than any of the other communities and utilized the same number of carbon sources as many of the other communities. These results demonstrate that community function (carbon source utilization) and community stability (resistance to invasion) are a function of the structural composition of the community irrespective of species richness or functional richness.
A bioluminescent reporter plasmid for naphthalene catabolism (pUTK21) was developed by transposon (Tn4431) insertion of the lux gene cassette from Vibrio fischeri into a naphthalene catabolic plasmid in Pseudomonas fluorescens. The insertion site of the lux transposon was the nahG gene encoding for salicylate hydroxylase. Luciferasemediated light production from P. fluorescens strains harboring this plasmid was induced on exposure to naphthalene or the regulatory inducer metabolite, salicylate. In continuous culture, light induction was rapid (15 minutes) and was highly responsive to dynamic changes in naphthalene exposure. Strains harboring pUTK21 were responsive to aromatic hydrocarbon contamination in Manufactured Gas Plant soils and produced sufficient light to serve as biosensors of naphthalene exposure and reporters of naphthalene biodegradative activity. The robust and sensitive nature of the bioluminescent reporter technology suggests that new sensing methods can be developed for on-line process monitoring and control in complex environmental matrices.
The promotion of polycyclic aromatic hydrocarbon (PAH) degradation was demonstrated in the rhizosphere of Festuca arundinacea with Pseudomonas fluorescens. P. fluorescens 5RL more significantly interacted with salicylate and dextrose in the agar containing tall fescue than agar without plant roots. Although the presence of tall fescue did not promote catabolic enzyme induction in the absence of salicylate, an increase in dioxygenase activity relative to no plant controls implies that this plant may enhance the degradation of PAHs or facilitate the genotypes that are capable of transforming PAH in the rhizosphere.
A low-power CMOS bioluminescent bioreporter integrated circuit (BBIC) is designed and fabricated for use in electronic/biological chemical sensing. The bioreporters are placed on a CMOS integrated circuit (IC) that detects bioluminescence, performs signal processing and produces a digital output pulse with a frequency that is proportional to the concentration of the target substance. The digital output pulse that contains the sensor information can then be transmitted to a remote location either wirelessly or via a data cable. The basic building blocks of the integrated circuit are the microluminometer and the transmitter. The microluminometer includes an integrated photodetector and a signal processor and is housed in a rugged inexpensive package that can be used in many remote applications in hazardous environmental monitoring. The total power consumption of the entire signal processing circuitry including the photodiodes is 3 mW from a 3.3-V power supply. This is lowered by a factor of 3 when compared to previous versions of the BBIC. In addition, it also integrates all features of detection, processing and data transmission into one small element. The bioreporter typically contains the luxCDABE reporter genes. The close proximity of the bioreporter and the sensing element eliminates the need for complex instrumentation to channel light from the bioreporters to the microluminometer. This paper presents an integrated CMOS microluminometer realized in 0.35-mum CMOS process and optimized for the detection of low-level bioluminescence as part of the BBIC. A flow-through test system was designed to expose the BBIC system composed of the microluminometer and the bioreporter Pseudomonas fluorescens 5RL to salicylate for determination of analytical benchmark data. The results obtained from the experiment are currently being used to study enclosures and micro-environment configurations for field-deployable BBICs for environmental monitoring
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