Table 1.
Comparison of low-cost fluorescence detectors.
All parameters are noted as specified by the authors, or were estimated, as indicated by ‘≈’ or ‘n.s.’ (not specified) if not enough information was available. Other abbreviations, ‘x’: available detection channels, ‘HV’: high voltage supply needed for capillary electrophoresis.
Fig 1.
(a) Photograph of the fully assembled detector (left) comprised of a detection unit and an assay cartridge (right). (b) Schematic of the experimental work flow. After sample deposition onto filter paper, the sample cartridge is clipped together and inserted into the detection unit. An automated time lapse measurement is then performed by the accompanying software.
Fig 2.
(a) The detector body includes the electronics and two flexible levers that extend to the LED and LDR. (b) The assay cartridge is made from two identical sides, each covered with lighting filter foils between which the filter paper containing the sample is placed, protected by two cover slides. (c) Sketch of the assembled detector. (d) Schematic illustrating the light path and electronic modules of the detector.
Fig 3.
Plastic filter foils for green fluorescence detection and calibration procedure.
(a) Excitation and (b) emission spectra of fluorescein (green) overlaid with the corresponding filter foil transmission spectra. The grey area in a) indicates the interval spanning 95% of the LED intensity. The combination of two blue and one orange filter foils allows for sufficient transmission of the excitation and emission light respectively, while blocking nearly all bleed-through excitation. (c) Calibration of the measured relative resistance for different dilutions of fluorescein over 3 replicates and 2 measurements per replicate. (d) Relative measurement uncertainty calculated as a function of the fluorescein concentration. The confident measurement range is taken as the range where the relative uncertainty is below 15%.
Fig 4.
(a) The user interface of the software allows to perform a calibration (to determine the dependence of the measured resistance on fluorophore concentration) or directly measure a sample. (b) Several parameters can be adjusted, such as the calibration parameters and the frequency of data acquisition. c) During measurements, acquired data can be plotted in real time.
Fig 5.
Detection of the transcription of a fluorescent aptamer.
(a) Scheme of the transcription of iSpinach aptamer, which binds the fluorophore DFHBI and increases its fluorescence. (b) Measurement of the concentration of iSpinach:DFHBI complex during transcription on the detector and in bulk (inset), with various concentrations of DNA template: 0 nM (grey), 25 nM (light blue), 50 nM (median blue) and 100 nM (dark blue). Thick line and shaded area represent respectively mean and measurement uncertainty as computed in S1 Appendix.
Fig 6.
(a) Mechanism of action of Cas13a: the protein (blue) forms a complex with a crRNA (grey) that consists of a Cas13a-handle and a sequence complementary to the target RNA (orange). Upon binding of the target RNA to the Cas13a-crRNA complex, Cas13a undergoes a conformational change that activates promiscuous RNase activity: Cas13a becomes an unspecific RNase [31]. (b) Mechanism of detection of Cas13a activity: a short RNA strand is modified with a fluorophore and a quencher. Upon cleavage by Cas13a, the fluorophore is released from the proximity of the quencher, and therefore fluorescence increases. (c-d) Measurement of Cas13a activity with 100 nM target RNA on paper in the detector (c) or in bulk using a plate reader (d). Activity in presence of a cognate (i.e. complementary to the crRNA) RNA target is compared to a non-cognate target. Residual activity in the presence of non-cognate target is likely due to an unspecific activity of Cas13a. In (d), positive control in bulk contains RNase A. (e-f) Response of the assay to increasing concentrations of cognate or non-cognate RNA target, in the detector (e) or in bulk (f). The LOD and LOQ as calculated based on the mean and standard deviation of background measurements are shown with dotted lines. Thick line and shaded area (c) and dots and error bars (e) represent mean and measurement uncertainty as computed in S1 Appendix.