On demand delivery and analysis of single molecules on a programmable nanopore-optofluidic device

Rahman, M.; Stott, Matthew A.; Harrington, M.; Li, Y.; Sampad, Mohammad Julker Neyen; Lancaster, Laura; Yuzvinsky, T. D.; Noller, Harry F.; Hawkins, Aaron R.; Schmidt, Holger

doi:10.1038/s41467-019-11723-7

Cited by 25 publications

(34 citation statements)

References 31 publications

(37 reference statements)

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“…3) In Stage 2 bunching of multiple AMP/ATP or free AA molecules can occur during translocation, leading to a missed read. In [18] a missed read is simply rejected. Here rejection is not an option, the solution lies in performing multiple measurements after reversing the voltage each time.…”

Section: Discussionmentioning

confidence: 99%

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Label-free amino acid identification forde novoprotein sequencing via tRNA charging and current blockade in a nanopore

Sampath¹

2020

Preprint

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Label-free identification of amino acids (AAs) based on superspecific transfer RNAs (tRNAs) and amino-acyl tRNA synthetase (AARS) is explored. Molecules of a specific AA, a tRNA and its cognate AARS, and adenosine triphosphate (ATP) from a reservoir are confined to a micro-sized cavity by hydraulic pressure to counter the effects of diffusion. In this confined space a cognate tRNA in the cavity gets charged with AA and adenosine monophosphate (AMP) is released. The products are transferred to an electrolytic cell with a nanopore where AA, AMP, and ATP cause current blockades of different sizes. If an AMP-sized blockade occurs it means that a tRNA molecule has been charged with AA, this identifies AA; otherwise the procedure is repeated with a different tRNA and AARS until AA is identified. Unlike in most other nanopore-based methods, there is no need for precise measurement of current blockade levels. The efficacy of the procedure is assessed by simulating the movement of particles in a reservoir-cavity structure and analyzing the blockades that occur in the nanopore. In the former the probability of reactants escaping the cavity is found to be near 0. In the latter the probability of an error in identifying an AMP blockade is found to be less than 10% in the worst case (which occurs with the largest volume AA, namely Tryptophan). Detailed information and data are provided in a Supplementary File.

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Section: Discussionmentioning

confidence: 99%

“…This was modified in [15] for the analysis of long DNA strands. Other related structures are described in [16][17][18].…”

Section: Stage 1: Charging Of Trna With An Amino Acid (Aa)mentioning

confidence: 99%

Label-free amino acid identification forde novoprotein sequencing via tRNA charging and current blockade in a nanopore

Sampath¹

2020

Preprint

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“…A part of the fluorescence light is collected via the orthogonal LC SC waveguides (green arrow) and sent to a photodetector (after filtering), which produces a detection spike as shown in the inset of Figure 5c. The device has sufficient enough sensitivity to detect nucleic acids [275] and viruses [276,277] and has been employed for a pool of applications such as particle trapping and manipulation [278][279][280], optical filtering [281,282], particle sorting [283], atomic spectroscopy [284,285], surface-enhanced Raman spectroscopy (SERS) detection [286], etc. The group has also demonstrated simultaneous multiplexed detection of single viruses using their ARROW optofluidic devices by taking advantage of the interference within a multimode interferometer waveguide [276].…”

Section: On-chip Optical Detection Of Single Moleculesmentioning

confidence: 99%

A Critical Review on the Sensing, Control, and Manipulation of Single Molecules on Optofluidic Devices

Rahman

Islam

et al. 2022

Micromachines

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Single-molecule techniques have shifted the paradigm of biological measurements from ensemble measurements to probing individual molecules and propelled a rapid revolution in related fields. Compared to ensemble measurements of biomolecules, single-molecule techniques provide a breadth of information with a high spatial and temporal resolution at the molecular level. Usually, optical and electrical methods are two commonly employed methods for probing single molecules, and some platforms even offer the integration of these two methods such as optofluidics. The recent spark in technological advancement and the tremendous leap in fabrication techniques, microfluidics, and integrated optofluidics are paving the way toward low cost, chip-scale, portable, and point-of-care diagnostic and single-molecule analysis tools. This review provides the fundamentals and overview of commonly employed single-molecule methods including optical methods, electrical methods, force-based methods, combinatorial integrated methods, etc. In most single-molecule experiments, the ability to manipulate and exercise precise control over individual molecules plays a vital role, which sometimes defines the capabilities and limits of the operation. This review discusses different manipulation techniques including sorting and trapping individual particles. An insight into the control of single molecules is provided that mainly discusses the recent development of electrical control over single molecules. Overall, this review is designed to provide the fundamentals and recent advancements in different single-molecule techniques and their applications, with a special focus on the detection, manipulation, and control of single molecules on chip-scale devices.

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“…Accordingly, nanopores have been used for exploring RNA translocation dynamics [10], tRNA translocation kinetics [11] and to investigate the folding of RNA pseudoknot structures [12]. Nanopores have also been previously used to detect bacterial 50S ribosomal subunits and control their translocation [13], and recently Rahman et al, reported the programmable delivery of 70S ribosomes with a nanopore integrated in an optofluidic chip [14].…”

Section: Introductionmentioning

confidence: 99%

Ribosome fingerprinting with a solid-state nanopore

Raveendran

Leach

Hopes

et al. 2020

Preprint

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Nanopores hold great potential for the analysis of complex biological molecules at the single entity level. One particularly interesting macromolecular machine is the ribosome, responsible for translating mRNA into proteins. In this study, we use a solid-state nanopore to fingerprint 80S ribosomes and polysomes from a human neuronal cell line and, Drosophila melanogaster cultured cells and ovaries. Specifically, we show that the peak amplitude and dwell time characteristics of 80S ribosomes are distinct from polysomes and can be used to discriminate ribosomes from polysomes in mixed samples. Moreover, we are able to distinguish large polysomes, containing more than 7 ribosomes, from those containing 2-3 ribosomes, and demonstrate a correlation between polysome size and peak amplitude. This study highlights the application of solid-state nanopores as a rapid analytical tool for the detection and characterization of ribosomal complexes.

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On demand delivery and analysis of single molecules on a programmable nanopore-optofluidic device

Cited by 25 publications

References 31 publications

Label-free amino acid identification forde novoprotein sequencing via tRNA charging and current blockade in a nanopore

Label-free amino acid identification forde novoprotein sequencing via tRNA charging and current blockade in a nanopore

A Critical Review on the Sensing, Control, and Manipulation of Single Molecules on Optofluidic Devices

Ribosome fingerprinting with a solid-state nanopore

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