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Fast custom wavelet analysis technique for single molecule detection and identification.

ABSTRACT: Many sensors operate by detecting and identifying individual events in a time-dependent signal which is challenging if signals are weak and background noise is present. We introduce a powerful, fast, and robust signal analysis technique based on a massively parallel continuous wavelet transform (CWT) algorithm. The superiority of this approach is demonstrated with fluorescence signals from a chip-based, optofluidic single particle sensor. The technique is more accurate than simple peak-finding algorithms and several orders of magnitude faster than existing CWT methods, allowing for real-time data analysis during sensing for the first time. Performance is further increased by applying a custom wavelet to multi-peak signals as demonstrated using amplification-free detection of single bacterial DNAs. A 4x increase in detection rate, a 6x improved error rate, and the ability for extraction of experimental parameters are demonstrated. This cluster-based CWT analysis will enable high-performance, real-time sensing when signal-to-noise is hardware limited, for instance with low-cost sensors in point of care environments.

SUBMITTER: Ganjalizadeh V

PROVIDER: S-EPMC8873225 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Fast custom wavelet analysis technique for single molecule detection and identification.

Ganjalizadeh Vahid V Meena Gopikrishnan G GG Wall Thomas A TA Stott Matthew A MA Hawkins Aaron R AR Schmidt Holger H

Nature communications 20220224 1

Many sensors operate by detecting and identifying individual events in a time-dependent signal which is challenging if signals are weak and background noise is present. We introduce a powerful, fast, and robust signal analysis technique based on a massively parallel continuous wavelet transform (CWT) algorithm. The superiority of this approach is demonstrated with fluorescence signals from a chip-based, optofluidic single particle sensor. The technique is more accurate than simple peak-finding a ...[more]

PMID: 35210454

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Project description:We describe the theory, experiment, and analysis of three-color Förster resonance energy transfer (FRET) spectroscopy for probing conformational dynamics of a fast-folding protein, α3D. In three-color FRET, site-specific labeling of fluorophores is required to avoid ambiguity resulting from various species with different combinations of labeling positions. To this end, we first attached two dyes to a cysteine residue and an unnatural amino acid and then appended a cysteine residue to the C-terminus of the protein by the sortase-mediated ligation for attaching the third dye. To determine all three FRET efficiencies, we used alternating excitation of the donor and acceptor 1 with two picosecond-pulsed lasers. Since the folded and unfolded states are not distinguishable in binned fluorescence trajectories due to fast-folding on a millisecond time scale, we used a maximum likelihood method that analyzes photon trajectories without binning the data. The extracted kinetic parameters agree very well with the previously measured parameters for the same protein with two-color FRET, suggesting that the addition of the third fluorophore does not affect the folding dynamics of the protein. From the extracted fractions of acceptor photon counts, the FRET efficiencies for all three dye pairs were calculated after various corrections. They were compared with the FRET efficiencies obtained from the global analysis of two-color segments collected in the same experiment. The FRET efficiencies of the folded state from the three-color segments agree with those from the two-color segments, whereas the three-color and two-color FRET efficiencies of the unfolded state are different. This happens because fluctuations of all three interdye distances contribute to the FRET efficiency measured in three-color FRET. We show that this difference can be accounted for by using the Gaussian chain model for the unfolded state with the parameters obtained from the analysis of two-color segments. This result shows that three-color FRET provides additional information on the flexibility of molecules that cannot be obtained from a combination of two-color FRET experiments with three dye pairs. Using the delay times of photons from the laser pulse, fluorescence lifetimes were determined using the maximum likelihood analysis. The correlation between FRET efficiencies and lifetimes of the donor, acceptor 1, and acceptor 2 was visualized in two-dimensional FRET efficiency-lifetime histograms. These histograms can be used to demonstrate the presence of conformational dynamics in a protein.

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Fast custom wavelet analysis technique for single molecule detection and identification.

Publications

Fast custom wavelet analysis technique for single molecule detection and identification.

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