Abstract
We describe the integration of techniques and technologies to develop a Point-of-Care for molecular diagnosis PoC-MD, based on a fluorescence lifetime measurement. Our PoC-MD is a low-cost, simple, fast, and easy-to-use general-purpose platform, aimed at carrying out fast diagnostics test through label detection of a variety of biomarkers. It is based on a 1-D array of 10 ultra-sensitive Single-Photon Avalanche Diode (SPAD) detectors made in a 0.18 μm High-Voltage Complementary Metal Oxide Semiconductor (HV-CMOS) technology. A custom microfluidic polydimethylsiloxane cartridge to insert the sample is straightforwardly positioned on top of the SPAD array without any alignment procedure with the SPAD array. Moreover, the proximity between the sample and the gate-operated SPAD sensor makes unnecessary any lens or optical filters to detect the fluorescence for long lifetime fluorescent dyes, such as quantum dots. Additionally, the use of a low-cost laser diode as pulsed excitation source and a Field-Programmable Gate Array (FPGA) to implement the control and processing electronics, makes the device flexible and easy to adapt to the target label molecule by only changing the laser diode. Using this device, reliable and sensitive real-time proof-of-concept fluorescence lifetime measurement of quantum dot QdotTM 605 streptavidin conjugate is demonstrated.
Highlights
IntroductionFluorescence-based techniques have become among the most widely used methods in clinical analysis and biomedical diagnosis [1] and have been applied in the field of high-throughput biotechnology for Fluorescence Activated Cell Sorting (FACS) and High-Throughput Screening (HTS)thanks to their accuracy, sensitivity (single molecule detection), and targeted labelling of biological samples [2].In particular, fluorescence measurements can provide information on the specific molecular makeup of a sample, and on the local environment surrounding the fluorescence molecule or fluorophore (such as pH, ion concentrations, etc.), which give value to the fluorescence based techniques a powerful analysis tool [3,4,5,6]
Nowadays, fluorescence-based techniques have become among the most widely used methods in clinical analysis and biomedical diagnosis [1] and have been applied in the field of high-throughput biotechnology for Fluorescence Activated Cell Sorting (FACS) and High-Throughput Screening (HTS)thanks to their accuracy, sensitivity, and targeted labelling of biological samples [2].In particular, fluorescence measurements can provide information on the specific molecular makeup of a sample, and on the local environment surrounding the fluorescence molecule or fluorophore, which give value to the fluorescence based techniques a powerful analysis tool [3,4,5,6]
The device performance is determined by the main parameters characterizing individual Single-Photon Avalanche Diode (SPAD), i.e., noise through Dark Count Rate (DCR) and sensitivity through Photon Detection Probability (PDP) [60]
Summary
Fluorescence-based techniques have become among the most widely used methods in clinical analysis and biomedical diagnosis [1] and have been applied in the field of high-throughput biotechnology for Fluorescence Activated Cell Sorting (FACS) and High-Throughput Screening (HTS)thanks to their accuracy, sensitivity (single molecule detection), and targeted labelling of biological samples [2].In particular, fluorescence measurements can provide information on the specific molecular makeup of a sample, and on the local environment surrounding the fluorescence molecule or fluorophore (such as pH, ion concentrations, etc.), which give value to the fluorescence based techniques a powerful analysis tool [3,4,5,6]. Time-resolved fluorescence techniques can address the limitations of intensity-based measurements by time resolving the fluorescence decay or lifetime, which is an intrinsic molecular property, independent of the fluorophore concentration and the excitation intensity [7]. Sensors 2019, 19, 445 the specificity of the fluorescence measurement by time domain discrimination This allows for the differentiation of fluorophores with overlapping emission spectra but different lifetimes [8], as well as to discern the light of interest from the background light due to autofluorescence of the biological sample, which can distort quantitative intensity based measurements [9]
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