Exploiting Molecular Barcodes in High-Throughput Cellular Assays

Binan, Loïc; Drobetsky, Elliot; Costantino, Santiago

doi:10.1177/2472630318824337

Cited by 7 publications

(10 citation statements)

References 86 publications

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“…With an optimized molecular system and method of reading, 64-bit (~10 19 codes) or higher encoding is conceivable. This would be enough to cover realistic needs for molecular barcodes 1 , 2 , 13 . For comparison, the current MAC (media access control) system uses 48-bit addresses for the unique identification of all devices connected to the Internet.…”

Section: Resultsmentioning

confidence: 99%

“…In this work, we present a prototype of paramagnetically encodable molecular architecture, demonstrate the encoding and decoding principles, provide practical examples of information encoding, and outline the possible future capabilities. The number of unique codes that can be generated in this way from a few chemical building blocks is compatible with the foreseeable needs in drug discovery 1 , 2 and anti-counterfeiting 13 applications. This work provides an alternative direction toward programmable digital molecular information systems.…”

Section: Introductionmentioning

confidence: 94%

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Paramagnetic encoding of molecules

et al. 2022

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Contactless digital tags are increasingly penetrating into many areas of human activities. Digitalization of our environment requires an ever growing number of objects to be identified and tracked with machine-readable labels. Molecules offer immense potential to serve for this purpose, but our ability to write, read, and communicate molecular code with current technology remains limited. Here we show that magnetic patterns can be synthetically encoded into stable molecular scaffolds with paramagnetic lanthanide ions to write digital code into molecules and their mixtures. Owing to the directional character of magnetic susceptibility tensors, each sequence of lanthanides built into one molecule produces a unique magnetic outcome. Multiplexing of the encoded molecules provides a high number of codes that grows double-exponentially with the number of available paramagnetic ions. The codes are readable by nuclear magnetic resonance in the radiofrequency (RF) spectrum, analogously to the macroscopic technology of RF identification. A prototype molecular system capable of 16-bit (65,535 codes) encoding is presented. Future optimized systems can conceivably provide 64-bit (~10^19 codes) or higher encoding to cover the labelling needs in drug discovery, anti-counterfeiting and other areas.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 94%

Paramagnetic encoding of molecules

et al. 2022

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“…In fluorescence readouts for drug screening and cellular and molecular studies in general, multiplexing strategies are used to increase the number of different cells or molecules that can be simultaneously identified and analyzed in a sample. A common barcoding strategy is to use fluorophores with different emission bands. , However, spectral overlap in the emission bands and constraints in filters, dichroic mirrors, and excitation light sources in the instruments set a limit for how many color channels can be used in parallel. Even with the best fluorescence microscopes available, it is difficult to include more than six color channels .…”

Section: Introductionmentioning

confidence: 99%

Fluorescence Bar-Coding and Flowmetry Based on Dark State Transitions in Fluorescence Emitters

Sandberg,

Demirbay,

Kulkarni

et al. 2023

J. Phys. Chem. B

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Reversible dark state transitions in fluorophores represent a limiting factor in fluorescence-based ultrasensitive spectroscopy, are a necessary basis for fluorescence-based superresolution imaging, but may also offer additional, largely orthogonal fluorescence-based readout parameters. In this work, we analyzed the blinking kinetics of Cyanine5 (Cy5) as a barcoding feature distinguishing Cy5 from rhodamine fluorophores having largely overlapping emission spectra. First, fluorescence correlation spectroscopy (FCS) solution measurements on mixtures of free fluorophores and fluorophore-labeled small unilamellar vesicles (SUVs) showed that Cy5 could be readily distinguished from the rhodamines by its reversible, largely excitation-driven trans−cis isomerization. This was next confirmed by transient state (TRAST) spectroscopy measurements, determining the fluorophore dark state kinetics in a more robust manner, from how the time-averaged fluorescence intensity varies upon modulation of the applied excitation light. TRAST was then combined with wide-field imaging of live cells, whereby Cy5 and rhodamine fluorophores could be distinguished on a whole cell level as well as in spatially resolved, multiplexed images of the cells. Finally, we established a microfluidic TRAST concept and showed how different mixtures of free Cy5 and rhodamine fluorophores and corresponding fluorophore-labeled SUVs could be distinguished on-the-fly when passing through a microfluidic channel. In contrast to FCS, TRAST does not rely on single-molecule detection conditions or a high time resolution and is thus broadly applicable to different biological samples. Therefore, we expect that the bar-coding concept presented in this work can offer an additional useful strategy for fluorescence-based multiplexing that can be implemented on a broad range of both stationary and moving samples.

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“…High-throughput combinatorial experiments have enormous potential for rapidly identifying small molecules, biomaterials, or biologics with superior performance, especially where a biological response remains unexplored 8,[33][34][35][36][37][38] . Such combinatorial screens are necessary for therapeutics development for cancer, diabetes, and other diseases.…”

Section: Introductionmentioning

confidence: 99%

In vivo screening of hydrogel library using cellular barcoding identifies biomaterials that mitigate host immune responses and fibrosis

Mukherjee

Cheng

et al. 2022

Preprint

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Biomaterials induced host immune responses, and fibrotic overgrowth remains a major barrier to the long-term function of medical devices and biomaterial consisting of tissue grafts. Screening new biomaterials to identify anti-fibrotic formulation requires in vivo testing, which is challenging to multiplex and remains a significant obstacle to progress in this field. Herein, we synthesized a combinatorial chemically modified hydrogel library and developed a cellular barcoding method that enables high-throughput multiplexed in vivo screening of 20 formulations in a single mouse and 100 formulations in a single non-human primate. Our screening method consists of implanting a mixture of biomaterials and each barcoded with human umbilical vein epithelial cells (HUVEC) from different individual donors. Single nucleotide polymorphism (SNP) genotypes of the cells were utilized as readouts using next-generation sequencing (NGS) to pair the material identity with material performance. Screening of the library using a xenogeneic transplantation model identified three novel lead hydrogel formulations (Z4-A10, Z1-A3, and Z2-A19) with improved anti-fibrotic properties that enable long-term cell viability. Z4-A10 was used to encapsulate human islets and validated for long-term glycemic control in an STZ-induced C57BL/6J diabetic mouse model. Leads, Z1-A3 and B2-A17, were further validated as immunomodulating coatings for medical-grade catheters to prevent fibrosis and occlusion, highlighting the translation of our screening approach and findings to other medical devices. Our results suggest that the developed cellular barcoding method and in vivo multiplexed screening technique can be leveraged to identify biomaterials for a wide range of clinical applications. Significantly, our newly discovered leads can improve the long-term performance of medical devices and encapsulated cell-based therapeutics.

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Exploiting Molecular Barcodes in High-Throughput Cellular Assays

Cited by 7 publications

References 86 publications

Paramagnetic encoding of molecules

Paramagnetic encoding of molecules

Fluorescence Bar-Coding and Flowmetry Based on Dark State Transitions in Fluorescence Emitters

In vivo screening of hydrogel library using cellular barcoding identifies biomaterials that mitigate host immune responses and fibrosis

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