<i>In Situ</i> Rolling Circle Amplification Förster Resonance Energy Transfer (RCA-FRET) for Washing-Free Real-Time Single-Protein Imaging

Francés‐Soriano, Laura; Leino, Mattias; Santos, Marcelina Cardoso Dos; Kovács, Dániel; Borbas, K. Eszter; Söderberg, Ola; Hildebrandt, Niko

doi:10.1021/acs.analchem.0c04828

Cited by 23 publications

(15 citation statements)

References 45 publications

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“…In the identification of gonorrhea pathogens, secretion smear, pathogenic bacteria culture, and SAT are commonly used in clinical practice [15]. Direct smear examination is limited by sampling and has a low positive rate for lesions other than male acute urethritis secretions and has difficulty in identifying different strains of Neisseria [16]. The SAT is prone to contamination and false positives and requires high sample purity.…”

Section: Discussionmentioning

confidence: 99%

Application of Isothermal Signal Amplification Technique in the Etiological Diagnosis of Gonorrhea and Drug Resistance Gene Detection

Chen

Zhang

et al. 2022

Computational and Mathematical Methods in Medicine

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Background: Isothermal signal amplification technique is developed based on the rolling ring amplification mechanism of cyclic DNA molecules in nature. This technique plays an extremely beneficial role in gonorrhea pathogen identification and drug resistance gene detection. Aims: This study analyzes the isothermal signal amplification techniques in the etiological diagnosis of gonorrhea and drug resistance gene detection. Materials and Methods: Urethral, cervical secretion, or prostatic fluid samples from 322 cases of gonorrhea collected from January 2018 to December 2021 at the STD clinic of our hospital dermatology department were selected for direct smear examination and gonococcal culture examination; DNA was extracted from urethral, cervical secretion, or prostatic fluid samples and then used for pathogen identification by SAT assay and rolling loop nucleic acid amplification technique, smear examination and pathogen culture examination methods, SAT assay, and isothermal signal amplification technique for comparative sensitivity and specificity analysis. Results: The highest rate of gonorrhea positivity was for the urine rolling loop nucleic acid amplification technique, followed by the swab rolling loop nucleic acid amplification technique, and the lowest rate of gonorrhea positivity was for the urine SAT test. The difference in the positivity rate between the two urine testing methods was statistically significant ( P < 0.05 ). The highest sensitivity of the urine rolling loop nucleic acid amplification technique method for the detection of gonorrhea pathogens and the lowest sensitivity of the urine SAT method were statistically significant ( P < 0.01 ). The differences in sensitivity and specificity between the swab rolling loop nucleic acid amplification technique and the swab SAT method were not statistically significant ( P > 0.05 ). ROC curves were plotted based on sensitivity and specificity, with swab SAT assay ( A U C = 0.998 ) > rolling loop nucleic acid amplification technique ( A U C = 0.981 ). Comparing the negative rates of urine and swab rolling loop nucleic acid amplification technique and urine SAT assay, the differences were not statistically significant ( P > 0.05 ). Conclusion: The isothermal signal amplification technique improves the shortcomings of gonorrhea pathogen identification means and drug resistance gene detection methods, with good detection sensitivity and specificity, simple operation, low price, and easy promotion, which has obvious advantages in clinical applications and epidemiological studies.

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Section: Discussionmentioning

confidence: 99%

Application of Isothermal Signal Amplification Technique in the Etiological Diagnosis of Gonorrhea and Drug Resistance Gene Detection

Chen

Zhang

et al. 2022

Computational and Mathematical Methods in Medicine

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“…Further improvements in image analysis, such as 3D analysis, will likely reduce or eliminate this issue. In addition, to refine the results, there is also a possibility to use FRET 52 between the different fluorophores on detection oligonucleotides A and B to determine if they are situated within the same RCP or not.…”

Section: Discussionmentioning

confidence: 99%

A method for Boolean analysis of protein interactions at a molecular level

et al. 2022

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Determining the levels of protein–protein interactions is essential for the analysis of signaling within the cell, characterization of mutation effects, protein function and activation in health and disease, among others. Herein, we describe MolBoolean – a method to detect interactions between endogenous proteins in various subcellular compartments, utilizing antibody-DNA conjugates for identification and signal amplification. In contrast to proximity ligation assays, MolBoolean simultaneously indicates the relative abundances of protein A and B not interacting with each other, as well as the pool of A and B proteins that are proximal enough to be considered an AB complex. MolBoolean is applicable both in fixed cells and tissue sections. The specific and quantifiable data that the method generates provide opportunities for both diagnostic use and medical research.

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“…56 For example, intracellular actin could be detected at the single-protein level via in situ RCA-TG-FRET coupled to immunochemistry (Figure 9A). 57 Because TG-FRET could occur only in the amplified RCP (hybridization of both Tb-DNA and dye-DNA), the signal per actin was both strongly amplified and very specific, thereby not interfering with background signals and avoiding any washing steps (Figure 9B). Wash-free and autofluorescence-free imaging with simple sample preparation and the capability for higher-order multiplexing has the potential to become very useful for sensitive imaging of intra-and extracellular biomarkers in clinical diagnostics.…”

Section: Tg-fret Imagingmentioning

confidence: 99%

Multiplexed Biosensing and Bioimaging Using Lanthanide-Based Time-Gated Förster Resonance Energy Transfer

Qiu

Santos

et al. 2022

Acc. Chem. Res.

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Metrics & MoreArticle Recommendations * sı Supporting Information CONSPECTUS:The necessity to scrutinize more and more biological molecules and interactions both in solution and on the cellular level has led to an increasing demand for sensitive and specific multiplexed diagnostic analysis. Photoluminescence (PL) detection is ideally suited for multiplexed biosensing and bioimaging because it is rapid and sensitive and there is an almost unlimited choice of fluorophores that provide a large versatility of photophysical properties, including PL intensities, spectra, and lifetimes.The most frequently used technique to detect multiple parameters from a single sample is spectral (or color) multiplexing with different fluorophores, such as organic dyes, fluorescent proteins, quantum dots, or lanthanide nanoparticles and complexes. In conventional PL biosensing approaches, each fluorophore requires a distinct detection channel and excitation wavelength. This drawback can be overcome by Forster resonance energy transfer (FRET) from lanthanide donors to other fluorophore acceptors. The lanthanides' multiple and spectrally narrow emission bands over a broad spectral range can overlap with several different acceptors at once, thereby allowing FRET from one donor to multiple acceptors. The lanthanides' extremely long PL lifetimes provide two important features. First, time-gated (TG) detection allows for efficient suppression of background fluorescence from the biological environment or directly excited acceptors. Second, temporal multiplexing, for which the PL lifetimes are adjusted by the interaction with the FRET acceptor, can be used to determine specific biomolecules and/or their conformation via distinct PL decays. The high signal-to-background ratios, reproducible and precise ratiometric and homogeneous (washing-free) sensing formats, and higher-order multiplexing capabilities of lanthanide-based TG-FRET have resulted in significant advances in the analysis of biomolecular recognition. Applications range from fundamental analysis of biomolecular interactions and conformations to high-throughput and point-of-care in vitro diagnostics and DNA sequencing to advanced optical encoding, using both liquid and solid samples and in situ, in vitro, and in vivo detection with high sensitivity and selectivity.In this Account, we discuss recent advances in lanthanide-based TG-FRET for the development and application of advanced immunoassays, nucleic acid sensing, and fluorescence imaging. In addition to the different spectral and temporal multiplexing approaches, we highlight the importance of the careful design and combination of different biological, organic, and inorganic molecules and nanomaterials for an adjustable FRET donor−acceptor distance that determines the ultimate performance of the diagnostic assays and conformational sensors in their physiological environment. We conclude by sharing our vision on how progress in the development of new sensing concepts, material combinations, and instrumentation can further advance ...

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In Situ Rolling Circle Amplification Förster Resonance Energy Transfer (RCA-FRET) for Washing-Free Real-Time Single-Protein Imaging

Cited by 23 publications

References 45 publications

Application of Isothermal Signal Amplification Technique in the Etiological Diagnosis of Gonorrhea and Drug Resistance Gene Detection

Application of Isothermal Signal Amplification Technique in the Etiological Diagnosis of Gonorrhea and Drug Resistance Gene Detection

A method for Boolean analysis of protein interactions at a molecular level

Multiplexed Biosensing and Bioimaging Using Lanthanide-Based Time-Gated Förster Resonance Energy Transfer

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