Name: Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers (Video) | JoVE Journal
Uploaded: 2025-03-21T15:00:00.000+00:00
Duration: 9 min 33 s
Description: Learn plasmonic nanotweezers for real-time monitoring of label-free protein conformational dynamics at the single-molecule level. This method enables investigation of binding kinetics and free energy landscapes without fluorophore modifications.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

S.Z. acknowledges support from the Biotechnology and Biological Sciences Research Council Doctoral Training Partnership (BBSRC DTP) (BB/T0083690/1). The authors acknowledge funding from the UK-India Education and Research Initiative (UKIERI). M.R. appreciates the support from the Royal Society and the Wolfson Foundation.

<ol><li>Peter Lu, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sizing+up+single-molecule+enzymatic+conformational+dynamics%5BTitle%5D)+AND+%22Chem+Soc+Rev%22%5BJournal%5D)">Sizing up single-molecule enzymatic conformational dynamics.</a> Chem Soc Rev. 43 (4), 1118-1143 (2014).</li>
<li>Mazal, H., Haran, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-molecule+FRET+methods+to+study+the+dynamics+of+proteins+at+work%5BTitle%5D)+AND+%22Curr+Opin+Biomed+Eng%22%5BJournal%5D)">Single-molecule FRET methods to study the dynamics of proteins at work.</a> Curr Opin Biomed Eng. 12, 8-17 (2019).</li>
<li>Ritchie, D. B., Woodside, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Probing+the+structural+dynamics+of+proteins+and+nucleic+acids+with+optical+tweezers%5BTitle%5D)+AND+%22Curr+Opin+Str+Biol%22%5BJournal%5D)">Probing the structural dynamics of proteins and nucleic acids with optical tweezers.</a> Curr Opin Str Biol. 34, 43-51 (2015).</li>
<li>Neupane, K., Solanki, A., Sosova, I., Belov, M., Woodside, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diverse+metastable+structures+formed+by+small+oligomers+of+%CE%B1-Synuclein+probed+by+force+spectroscopy%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Diverse metastable structures formed by small oligomers of α-Synuclein probed by force spectroscopy.</a> PLoS One. 9 (1), e86495(2014).</li>
<li>Hughes, M. L., Dougan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+physics+of+pulling+polyproteins%3A+a+review+of+single+molecule+force+spectroscopy+using+the+AFM+to+study+protein+unfolding%5BTitle%5D)+AND+%22Rep+Prog+Phys%22%5BJournal%5D)">The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding.</a> Rep Prog Phys. 79 (7), 076601-076601 (2016).</li>
<li>Young, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+mass+imaging+of+single+biological+macromolecules%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Quantitative mass imaging of single biological macromolecules.</a> Science. 360 (6387), 423-427 (2018).</li>
<li>Schmid, S., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanopores%3A+a+versatile+tool+to+study+protein+dynamics%5BTitle%5D)+AND+%22Essays+Biochem%22%5BJournal%5D)">Nanopores: a versatile tool to study protein dynamics.</a> Essays Biochem. 65 (1), 93-107 (2021).</li>
<li>Van Meervelt, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+conformational+changes+and+controlled+orientation+of+native+proteins+inside+a+protein+nanoreactor%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">Real-time conformational changes and controlled orientation of native proteins inside a protein nanoreactor.</a> J Am Chem Soc. 139 (51), 18640-18646 (2017).</li>
<li>Awasthi, S., Ying, C., Li, J., Mayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+determination+of+the+size+and+shape+of+single+%CE%B1-Synuclein+oligomers+in+solution%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">Simultaneous determination of the size and shape of single α-Synuclein oligomers in solution.</a> ACS Nano. 17 (13), 12325-12335 (2023).</li>
<li>Sµnchez-Rico, C., Von Vithenberg, L., Warner, L., Lamb, D. C., Sattler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+fluorophore+attachment+on+protein+conformation+and+dynamics+studied+by+spFRET+and+NMR+spectroscopy%5BTitle%5D)+AND+%22Chemistry%22%5BJournal%5D)">Effects of fluorophore attachment on protein conformation and dynamics studied by spFRET and NMR spectroscopy.</a> Chemistry. 23 (57), 14267-14277 (2017).</li>
<li>Yin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+does+fluorescent+labeling+affect+the+binding+kinetics+of+proteins+with+intact+cells%5BTitle%5D)+AND+%22Biosens+Bioelectr%22%5BJournal%5D)">How does fluorescent labeling affect the binding kinetics of proteins with intact cells.</a> Biosens Bioelectr. 66, 412-416 (2015).</li>
<li>Berkovich, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rate+limit+of+protein+elastic+response+is+tether+dependent%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci%22%5BJournal%5D)">Rate limit of protein elastic response is tether dependent.</a> Proc Natl Acad Sci. 109 (36), 14416-14421 (2012).</li>
<li>Dahmardeh, M., Mirzaalian Dastjerdi, H., Mazal, H., Köstler, H., Sandoghdar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-supervised+machine+learning+pushes+the+sensitivity+limit+in+label-free+detection+of+single+proteins+below+10+kDa%5BTitle%5D)+AND+%22Nat+Meth%22%5BJournal%5D)">Self-supervised machine learning pushes the sensitivity limit in label-free detection of single proteins below 10 kDa.</a> Nat Meth. 20 (3), 442-447 (2023).</li>
<li>Becker, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+quantitative+description+for+optical+mass+measurement+of+single+biomolecules%5BTitle%5D)+AND+%22ACS+Photon%22%5BJournal%5D)">A quantitative description for optical mass measurement of single biomolecules.</a> ACS Photon. 10 (8), 2699-2710 (2023).</li>
<li>Houghtaling, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estimation+of+shape%2C+volume%2C+and+dipole+moment+of+individual+proteins+freely+transiting+a+synthetic+nanopore%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">Estimation of shape, volume, and dipole moment of individual proteins freely transiting a synthetic nanopore.</a> ACS Nano. 13 (5), 5231-5242 (2019).</li>
<li>Plesa, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fast+translocation+of+proteins+through+solid+state+nanopores%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Fast translocation of proteins through solid state nanopores.</a> Nano Lett. 13 (2), 658-663 (2013).</li>
<li>Schmid, S., Stömmer, P., Dietz, H., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanopore+electro-osmotic+trap+for+the+label-free+study+of+single+proteins+and+their+conformations%5BTitle%5D)+AND+%22Nat+Nanotechnol%22%5BJournal%5D)">Nanopore electro-osmotic trap for the label-free study of single proteins and their conformations.</a> Nat Nanotechnol. 16 (11), 1244-1250 (2021).</li>
<li>Shi, X., Verschueren, D. V., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Active+delivery+of+single+DNA+molecules+into+a+plasmonic+nanopore+for+label-free+optical+sensing%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Active delivery of single DNA molecules into a plasmonic nanopore for label-free optical sensing.</a> Nano Lett. 18 (12), 8003-8010 (2018).</li>
<li>Verschueren, D. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-free+optical+detection+of+DNA+translocations+through+plasmonic+nanopores%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">Label-free optical detection of DNA translocations through plasmonic nanopores.</a> ACS Nano. 13 (1), 61-70 (2019).</li>
<li>Verschueren, D., Shi, X., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nano-optical+tweezing+of+single+proteins+in+plasmonic+nanopores%5BTitle%5D)+AND+%22Small+Meth%22%5BJournal%5D)">Nano-optical tweezing of single proteins in plasmonic nanopores.</a> Small Meth. 3 (5), 1800465(2019).</li>
<li>Peri, S. S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+low+affinity+binding+interactions+between+natural+killer+cell+inhibitory+receptors+and+targeting+ligands+with+a+self-induced+back-action+actuated+nanopore+electrophoresis+%28SANE%29+sensor%5BTitle%5D)+AND+%22Nanotechnology%22%5BJournal%5D)">Quantification of low affinity binding interactions between natural killer cell inhibitory receptors and targeting ligands with a self-induced back-action actuated nanopore electrophoresis (SANE) sensor.</a> Nanotechnology. 32 (4), 045501(2020).</li>
<li>Leong, I. W., Tsutsui, M., Yokota, K., Taniguchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Salt+gradient+control+of+translocation+dynamics+in+a+solid-state+nanopore%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Salt gradient control of translocation dynamics in a solid-state nanopore.</a> Anal Chem. 93 (49), 16700-16708 (2021).</li>
<li>Juan, M. L., Righini, M., Quidant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasmon+nano-optical+tweezers%5BTitle%5D)+AND+%22Nat+Photon%22%5BJournal%5D)">Plasmon nano-optical tweezers.</a> Nat Photon. 5 (6), 349-356 (2011).</li>
<li>Juan, M. L., Gordon, R., Pang, Y., Eftekhari, F., Quidant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-induced+back-action+optical+trapping+of+dielectric+nanoparticles%5BTitle%5D)+AND+%22Nat+Phys%22%5BJournal%5D)">Self-induced back-action optical trapping of dielectric nanoparticles.</a> Nat Phys. 5 (12), 915-919 (2009).</li>
<li>Pang, Y., Gordon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+trapping+of+12+nm+dielectric+spheres+using+double-nanoholes+in+a+gold+film%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Optical trapping of 12 nm dielectric spheres using double-nanoholes in a gold film.</a> Nano Lett. 11 (9), 3763-3767 (2011).</li>
<li>Pang, Y., Gordon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+trapping+of+a+single+protein%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Optical trapping of a single protein.</a> Nano Lett. 12 (1), 402-406 (2012).</li>
<li>Kotnala, A., Gordon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+high-efficiency+trapping+of+nanoparticles+in+a+double+nanohole+optical+tweezer%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Quantification of high-efficiency trapping of nanoparticles in a double nanohole optical tweezer.</a> Nano Lett. 14 (2), 853-856 (2014).</li>
<li>Booth, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modelling+of+the+dynamic+polarizability+of+macromolecules+for+single-molecule+optical+biosensing%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Modelling of the dynamic polarizability of macromolecules for single-molecule optical biosensing.</a> Sci Rep. 12 (1), 1995(2022).</li>
<li>Yousefi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+monitoring+of+in+situ+Iron+loading+into+single%2C+native+ferritin+proteins%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Optical monitoring of in situ Iron loading into single, native ferritin proteins.</a> Nano Lett. 23 (8), 3251-3258 (2023).</li>
<li>Yousefi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+Flexibility+and+Disassembly+Kinetics+of+Single+Ferritin+Molecules+Using+Optical+Nanotweezers%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">Structural Flexibility and Disassembly Kinetics of Single Ferritin Molecules Using Optical Nanotweezers.</a> ACS Nano. 18 (24), 15617-15626 (2024).</li>
<li>Peters, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Energy+landscape+of+conformational+changes+for+a+single+unmodified+protein%5BTitle%5D)+AND+%22NPJ+Biosens%22%5BJournal%5D)">Energy landscape of conformational changes for a single unmodified protein.</a> NPJ Biosens. 1 (1), 14-14 (2024).</li>
<li>Peters, M., McIntosh, D., Branzan Albu, A., Ying, C., Gordon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-free+tracking+of+proteins+through+plasmon-enhanced+interference%5BTitle%5D)+AND+%22ACS+Nanosci+Au%22%5BJournal%5D)">Label-free tracking of proteins through plasmon-enhanced interference.</a> ACS Nanosci Au. 4 (1), 69-75 (2024).</li>
<li>Ying, C., et al. Watching single unmodified enzymes at work. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aYing%2C+C+%22arXiv%22">arXiv</a>. , (2021).</li>
<li>Yang-Schulz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+observation+of+small+molecule+activator+binding+to+single+PR65+protein%5BTitle%5D)+AND+%22NPJ+Biosensing%22%5BJournal%5D)">Direct observation of small molecule activator binding to single PR65 protein.</a> NPJ Biosensing. 2 (1), 1-10 (2025).</li>
<li>Yang, W., van Dijk, M., Primavera, C., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FIB-milled+plasmonic+nanoapertures+allow+for+long+trapping+times+of+individual+proteins%5BTitle%5D)+AND+%22iScience%22%5BJournal%5D)">FIB-milled plasmonic nanoapertures allow for long trapping times of individual proteins.</a> iScience. 24 (11), 103237(2021).</li>
<li>Koya, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+plasmonic+nanocavities+for+optical+trapping-assisted+biosensing+applications%5BTitle%5D)+AND+%22Adv+Optical+Mater%22%5BJournal%5D)">Novel plasmonic nanocavities for optical trapping-assisted biosensing applications.</a> Adv Optical Mater. 8 (7), 1901481(2020).</li>
<li>Goswami, A., Umashankar, R., Gupta, A. K., Aravindan, S., Rao, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+microstructured+surface+using+the+FIB%5BTitle%5D)+AND+%22J+Micromanufact%22%5BJournal%5D)">Development of a microstructured surface using the FIB.</a> J Micromanufact. 1 (1), 53-61 (2018).</li>
<li>Onuta, T. D., Waegele, M., DuFort, C. C., Schaich, W. L., Dragnea, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+field+enhancement+at+cusps+between+adjacent+nanoapertures%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Optical field enhancement at cusps between adjacent nanoapertures.</a> Nano Lett. 7 (3), 557-564 (2007).</li>
<li>Al-Ani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+PEG-thiol+derivatives+on+controlling+cellular+and+bacterial+interactions+with+gold+surfaces%5BTitle%5D)+AND+%22Appl+Surf+Sci%22%5BJournal%5D)">The influence of PEG-thiol derivatives on controlling cellular and bacterial interactions with gold surfaces.</a> Appl Surf Sci. 462, 980-990 (2018).</li>
<li>Schuler, B., Hofmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-molecule+spectroscopy+of+protein+folding+dynamics-expanding+scope+and+timescales%5BTitle%5D)+AND+%22Curr+Opin+Str+Biol%22%5BJournal%5D)">Single-molecule spectroscopy of protein folding dynamics-expanding scope and timescales.</a> Curr Opin Str Biol. 23 (1), 36-47 (2013).</li>
<li>Kopac, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+corona%2C+understanding+the+nanoparticle-protein+interactions+and+future+perspectives%3A+A+critical+review%5BTitle%5D)+AND+%22Int+J+Biol+Macromol%22%5BJournal%5D)">Protein corona, understanding the nanoparticle-protein interactions and future perspectives: A critical review.</a> Int J Biol Macromol. 169, 290-301 (2021).</li>
<li>Li, X., Guo, W., Xu, R., Song, Z., Ni, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+interaction+mechanism+between+gold+nanoparticles+and+proteins%3A+Lysozyme%2C+trypsin%2C+pepsin%2C+%CE%B3-globulin%2C+and+hemoglobin%5BTitle%5D)+AND+%22Spectrochim+Acta+A+Mol+Biomol+Spectrosc%22%5BJournal%5D)">The interaction mechanism between gold nanoparticles and proteins: Lysozyme, trypsin, pepsin, γ-globulin, and hemoglobin.</a> Spectrochim Acta A Mol Biomol Spectrosc. 272, 120983(2022).</li>
<li>Emilsson, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strongly+stretched+protein+resistant+poly%28ethylene+glycol%29+brushes+prepared+by+grafting-To%5BTitle%5D)+AND+%22ACS+Appl+Mater+Inter%22%5BJournal%5D)">Strongly stretched protein resistant poly(ethylene glycol) brushes prepared by grafting-To.</a> ACS Appl Mater Inter. 7 (14), 7505-7515 (2015).</li>
<li>Theil, E. C., Liu, X. S., Tosha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gated+pores+in+the+ferritin+protein+nanocage%5BTitle%5D)+AND+%22Inorg+Chim+Acta%22%5BJournal%5D)">Gated pores in the ferritin protein nanocage.</a> Inorg Chim Acta. 361 (4), 868-874 (2008).</li>
<li>Theil, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ferritin+iron+entry+and+exit+problem%5BTitle%5D)+AND+%22Inorg+Chim+Acta%22%5BJournal%5D)">The ferritin iron entry and exit problem.</a> Inorg Chim Acta. 297 (1), 242-251 (2000).</li>
<li>Takahashi, T., Kuyucak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+properties+of+threefold+and+fourfold+channels+in+Ferritin+D%3Ddeduced+from+electrostatic+calculations%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Functional properties of threefold and fourfold channels in Ferritin D=deduced from electrostatic calculations.</a> Biophys J. 84 (4), 2256-2263 (2003).</li>
<li>Gemeinhardt, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-free+imaging+of+single+proteins+secreted+from+living+cells+via+iSCAT+microscopy%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Label-free imaging of single proteins secreted from living cells via iSCAT microscopy.</a> J Vis Exp. (141), e58486(2018).</li>
<li>Piliarik, M., Sandoghdar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+optical+sensing+of+single+unlabelled+proteins+and+super-resolution+imaging+of+their+binding+sites%5BTitle%5D)+AND+%22Nat+Comm%22%5BJournal%5D)">Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites.</a> Nat Comm. 5 (1), 4495(2014).</li>
</ol>

The authors have nothing to disclose.

A crucial step in the protocol is ensuring the flow cell does not leak before being set into the stage, which should be tested outside of the mount at a high flow rate beforehand. Leakage after the sample is mounted can damage optical components, in particular the bottom objective. 
The alignment may drift from its optimal position over time during an experiment, causing signal variation due to the sensitivity of the plasmonic nanotweezers. When this occurs, realign using the piezoelectric controls to maximum transmission and minimum standard deviation of the baseline, as a noisy baseline reduces the quality of data. Care should be taken to make meticulous notes of when alignments are made to remove the risk of confusing user interference for a trapping event. If major drifting occurs, gently align the stage to minimize the risk of releasing the protein and note the time of adjustment.
Modifications and alterations to the presented technique can be made based on specific experimental needs. For example, a temperature-controlled stage can help cool/heat the sample as desired instead of using laser heating to increase the temperature31,33. Other techniques, for example, interferometric scattering microscopy (iSCAT), can provide interference of the protein within the scattering field of the DNH, obtaining additional signal proportional to the polarizability of the protein, which is associated with particle size47,48.
Plasmonic nanotweezers are purely a temporal sensing technique as data is recorded by the APD (a single-pixel detector). This technique provides no direct information on the structural changes of a protein, such as which regions of the protein are involved in a conformational change or where a ligand or another protein may bind to the protein. Additionally, the technique is limited to a temporal range of &gt;1 &#181;s due to the sampling rate of the data acquisition card (1 MHz). Considering the Nyquist frequency, where the highest possible range is half the acquisition rate, in this case, 2 &#181;s under perfect conditions.
In this protocol, we have described the process of setting up a plasmonic nanotweezers experiment to trap a single protein and monitor changes in its conformational dynamics over time. The technique can be developed on any home-built or commercial microscope. Contrary to fluorescence or tethering-based approaches, such as smFRET and optical tweezers, the technique can trap proteins without labels or tethers and still achieve single-molecule sensitivity. The solution conditions can be changed whilst the protein is trapped using a microfluidics system, allowing for real-time monitoring of the effect of different solutions on the protein. These features make plasmonic nanotweezers a promising tool within the biophysics and biosensing fields, particularly for proteins that conventional techniques struggle to interrogate in their native states. Future applications will focus on decoding the conformational dynamics of more dynamic proteins such as intrinsically disordered proteins and proteins containing intrinsically disordered regions, and membrane proteins, whose dynamics and even structure elude current techniques.

Current single-molecule techniques for interrogating protein conformational dynamics include labelling-based methods such as fluorescence resonance energy transfer (FRET)1,2, tethering-based approaches such as optical tweezers3,4and atomic force microscopy (AFM)5, interference-based techniques such as interference scattering microscopy (iSCAT)6, or nanofluidic-based techniques such as nanopores7,8,9. While these methods have many advantages; a few key limitations prevent them from providing data on unmodified protein conformational dynamics. FRET and optical tweezers require fluorophore labeling or tethering to a surface, which may affect the proteins&#39; biophysical properties10,11,12. iSCAT, although technically label-free, also requires interaction between the protein and a surface to observe interference generated between the two which potentially affects the proteins&#39; properties. Moreover, limited by its signal-to-noise ratio, iSCAT can only detect proteins &#62;40 kDa due to equipment noise and speckle-like background fluctuations13. Although this size limit can be alleviated through machine learning, buffer components are limited as they can affect optical properties, resulting in noisy data13,14. Nanopores present fast translocation times of proteins through the pore (usually within 5 &#181;s), rendering them unable to detect slower conformational dynamics15,16, although research into alleviating these limitations, such as the use of DNA origami in a nanopore electro-osmotic trap17 or incorporation of plasmonics18,19,20,21. Additionally, high salt concentrations, typically around 1 M, can reduce the applicability of the data for in vivo work15,22. The ideal single-molecule technique for protein characterization should monitor proteins in real time and capture conformational dynamics over longer durations (i.e., milliseconds) without the need for modifications to the protein or non-native solution conditions.
Plasmonic nanotweezers are similar to conventional optical tweezers, in the sense that they use light to trap matter. Plasmonic nanotweezers, however, utilize localized surface plasmonic resonance (LSPR) to enhance the electric field by several orders of magnitude to generate a gradient force strong enough to trap single nanoparticles23. Additionally, the trapped particle plays an active role in enhancing the strength of the trap, known as self-induced back-action (SIBA) trapping for nanoaperture structures24. This SIBA trapping allows for low laser powers (i.e., milliwatts) to trap small particles down to only a few nanometers in diameter, such as proteins25,26,27. Trapping single protein molecules within the hotspot region causes a shift in the local refractive index (nprotein &#62; nwater), altering light scattering based on the molecule&#39;s polarizability which is influenced by the protein&#39;s volume, shape anisotropy, and refractive index28. An avalanche photodiode (APD) then detects this information to monitor subsequent changes in light scattering. Moreover, plasmonic nanotweezers allow monitoring of the trapped proteins in real-time without labeling, tethers and harsh solution conditions for long time periods (i.e., minutes to hours)29, fulfilling the criteria for an ideal single-molecule technique for proteins. Using a double nanohole (DNH) structure, plasmonic nanotweezers have demonstrated their ability to trap various proteins and elucidate key information from them including conformational transitions29, disassembly kinetics30, energy landscapes31, diffusion tracking32, and ligand binding33,34. Besides DNH structures, alternative structure geometries have been demonstrated to trap particles with small particle sizes35,36. In this protocol the fundamental steps to set up and run a plasmonic nanotweezers setup with an integrated microfluidics system are presented. We hope this protocol will help to increase the accessibility and understanding of plasmonic nanotweezers to researchers, particularly those in the structural biology and biophysics fields.

<table><tbody><tr><td>100x Objective (NA = 1.25)</td><td>Olympus</td><td>PLN100XO</td><td>Clean oil only with lens cleaning tissue in one motion</td></tr><tr><td>12-1 rotary bidirectional microfluidic valve</td><td>Elveflow</td><td></td><td></td></tr><tr><td>3/2 way solenoid valve</td><td>Elveflow</td><td></td><td></td></tr><tr><td>4x Objective (NA = 0.1)</td><td>Olympus</td><td>PLN4XP</td><td></td></tr><tr><td>Airgun</td><td>RS Components</td><td>666-6772</td><td></td></tr><tr><td>Avalanche photodiode (APD)</td><td>Thorlabs</td><td>APD120A/M</td><td>Do not oversaturate, 50 MHz bandwidth, static sensitive</td></tr><tr><td>Butterfly laser diode</td><td>Thorlabs</td><td>FPL852S</td><td></td></tr><tr><td>Charge-coupled device (camera)</td><td>Hikrobot</td><td>MV-CE200-10UC</td><td></td></tr><tr><td>Crystallising dish</td><td>VWR</td><td>216-0065</td><td></td></tr><tr><td>Data acquisition card&amp;nbsp;</td><td>National instruments</td><td>USB-6361</td><td>2 MHz sampling rate</td></tr><tr><td>Double-sided tape</td><td>Adhesive Research Inc</td><td>ARcare92712</td><td>Cut appropriate shape for flow cell with a laser cutter</td></tr><tr><td>Ethanol</td><td></td><td></td><td>Fume hood only, use gloves</td></tr><tr><td>Fibreport collimator</td><td>Thorlabs</td><td>PAF2-A7B</td><td></td></tr><tr><td>Glass microscope coverslips (thickness 0.17 mm)</td><td></td><td></td><td></td></tr><tr><td>Half wave plate</td><td>Thorlabs</td><td>AHWP10M-980</td><td></td></tr><tr><td>Ideal-tek 120 mm, stainless steel, straight tweezers</td><td>RS Components</td><td>282-7472</td><td>For holding sample and peeling double sided tape</td></tr><tr><td>Isopropanol (IPA)</td><td></td><td></td><td>Fume hood only, use gloves</td></tr><tr><td>Lens cleaning tissue</td><td>Thorlabs</td><td>MC-5</td><td></td></tr><tr><td>Metrosil rapid duplicating silicone parts A and B</td><td>Metrodent</td><td>MSILR/1&amp;nbsp;</td><td></td></tr><tr><td>Microscope slides (75 mm x 26 mm)</td><td></td><td></td><td></td></tr><tr><td>Modular Optical Tweezers System</td><td>Thorlabs</td><td>OTKB/M-CUSTOM</td><td></td></tr><tr><td>Objective oil</td><td>Olympus</td><td></td><td>Use gloves, store between 2&lt;sup&gt;o&lt;/sup&gt;C-8&lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>Photodiode laser mount</td><td>Thorlabs</td><td>CLD1015</td><td></td></tr><tr><td>Piezoelectric controller</td><td>Thorlabs</td><td>MDT693B</td><td></td></tr><tr><td>Piezoelectric stage</td><td>Thorlabs</td><td>Nanomax 300</td><td></td></tr><tr><td>Planoconcave lens (&lt;em&gt;f&lt;/em&gt; = -50 mm)</td><td>Thorlabs</td><td>LC1715-B</td><td></td></tr><tr><td>Planoconvex lens (&lt;em&gt;f&lt;/em&gt; = 150 mm)</td><td>Thorlabs</td><td>LA1433-B</td><td></td></tr><tr><td>Planoconvex lens (&lt;em&gt;f&lt;/em&gt; = 60 mm)</td><td>Thorlabs</td><td>LA1134-B</td><td></td></tr><tr><td>Poly(ethylene glycol) methyl ether thiol (PEG-thiol) 800 MW</td><td>Sigma Aldrich</td><td>729108</td><td>Store between 2&lt;sup&gt;o&lt;/sup&gt;C-8&lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>PTFE tubing (ID: 0.18 mm)</td><td>Vici Jour</td><td>JR-T-6805-M25</td><td></td></tr><tr><td>PTFE tubing (ID: 0.8 mm)</td><td>Cole-Parmer</td><td>WZ-21942-72</td><td></td></tr><tr><td>SEM-FIB</td><td>Zeiss</td><td>Crossbeam 550</td><td>Gallium ion source</td></tr><tr><td>Shortpass dichroic mirror 805 nm</td><td>Thorlabs</td><td>DMSP805</td><td></td></tr><tr><td>Silver mirror</td><td>Thorlabs</td><td>PF-10-03-P01</td><td></td></tr><tr><td>Syringe pump</td><td>Harvard Apparatus</td><td>70-4511</td><td></td></tr><tr><td>Weller Erem 120 mm stainless steel, flat, rounded tweezers</td><td>RS Components</td><td>176-1150</td><td>For holding glass coverslip and peeling glue from flow cell</td></tr></tbody></table>

monitoring conformational dynamics of single unmodified proteins using plasmonic nanotweezers

Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes can alter protein biophysics and reduce the usefulness of the data acquired. Plasmonic nanotweezers is a technique that uses localized surface plasmon resonance (LSPR) on gold nanostructures to enhance the electric field within a confined hotspot region. This field enhancement allows for the use of low laser powers to trap single nanoparticles far smaller than conventional optical tweezers, down to only a few nanometers in diameter, such as single proteins. Trapping of single protein molecules within the hotspot region induces a shift in the local refractive index (nprotein &#62; nwater), altering light scattering as a product of the molecule&#39;s polarisability, which is affected by its volume, shape anisotropy, and refractive index. An avalanche photodiode (APD) collects the subsequent changes in light scattering. These alterations can then be analyzed to determine changes in the trapped molecule, including its size, global conformation, and dynamics of conformational change over time. The incorporation of microfluidics within the system allows for controlled environmental changes and real-time monitoring of their subsequent effects on the molecule. In this protocol, we demonstrate the steps to trap single protein molecules, alter their environmental solution conditions, and monitor their corresponding conformational changes using a plasmonic nanotweezers system.

Following data acquisition, data analysis can be performed on the raw data using MATLAB code to generate traces from the raw data collected by the APD. Figure 3 depicts an exemplar trapping trace including the baseline before trapping, the trapping event where a large change in transmission (ΔT/T0) and standard deviation is observed before the laser is turned off for around 5s before being turned back on. A significant reduction in standard deviation and return of transmission to similar levels as the baseline indicates the release of protein. Linear drift is removed from the trace using the MATLAB function detrend.m, and then the mean value of the data is added back to the detrended trace. Occasionally, we need to detrend the trace as the setup drifts over time, causing a linear decrease in transmission (see the grey trace in Figure 3). Small changes in the baseline traces before and after trapping are due to stage adjustment to optimize the baseline with minimal standard deviation, demonstrated in Figure 4A. Sometimes, protein molecules are visible in the trace without being trapped, termed passing-by proteins. Proteins passing by appear as a sharp change in transmission, similar to a typical trap (Figure 4B), but with a significantly shorter duration, as shown in Figure 4A. Power spectral density (PSD) presents another analysis to confirm protein trapping by providing signal strength at various frequencies. Protein conformational motions are typically seen in the &gt;1 µs range by single molecule spectroscopy methods40. Figure 4C demonstrates that compared to the baseline, trapping a protein leads to higher signal strength, at least within the 10 kHz range (&gt; 100 µs). It also highlights the importance of aligning the stage to an optimized baseline, as a bad baseline could increase the noise at frequencies between 50-500 Hz, a frequency range overlaid with protein conformational motions.
<img alt="APD voltage graph showing protein release with signal filtering and detrending at multiple frequencies." class="xfigimg" src="/files/ftp_upload/68093/68093fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Full trapping trace for a single protein. Representative trace for a full trap, including the baseline, trapping a protein, and release of the protein. Jumps in trace before and after trapping are due to alignment. <a href="https://www.jove.com/files/ftp_upload/68093/68093fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Protein detection process through nanopore sensor; voltage signal graphs and frequency analysis chart." class="xfigimg" src="/files/ftp_upload/68093/68093fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Common trace events. (A) Examples of an alignment from a bad to a good baseline and a protein passing close to the hotspot. (B) Trapping trace showing the process from the baseline when the DNH hotspot is empty to when the protein is trapped. (C) Power spectral density (PSD) plot between the good and bad baselines depicted in (A) and the protein trapped in (B). Higher PSD values indicate greater noise at particular frequencies. <a href="https://www.jove.com/files/ftp_upload/68093/68093fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Most trapping events follow the same general pattern as the trace in Figure 3, although occasional issues may arise during experiments. For most experiments, the protein should be released manually by turning the laser off once the desired experiment is completed. In some cases, however, the protein can leave the trap without intervention, as shown in Figure 5A. Conversely, sometimes proteins can remain at the trapping site even after turning the laser off, likely due to the protein sticking to the sample. This sticking results in a noisy trace after turning the laser off and on (see Figure 5B). The likelihood of this occurring depends on the protein, as some proteins are more prone to surface adsorption41,42. The use of a coating such as PEG-thiol can reduce the chances of protein sticking39,43. Unless desired, such as studying protein-protein interactions, another issue is double trapping, where a second protein is trapped after the first trap. This is characterized by another sharp increase in transmission, similar to the first trap, and a change in standard deviation (see Figure 5C). 
<img alt="Voltage-time graph of protein trapping experiment; filtered signals indicate protein dynamics." class="xfigimg" src="/files/ftp_upload/68093/68093fig05.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: Examples of undesirable trapping events. (A) Unintended release of a protein from the DNH hotspot. (B) Example of protein becoming stuck on the sample surface in the DNH hotspot. (C) Trace jump occurs when a second protein is trapped whilst the first still remains in the DNH hotspot. <a href="https://www.jove.com/files/ftp_upload/68093/68093fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A representative experiment carried out on in situ iron loading to an apo-ferritin molecule demonstrates the use of plasmonic nanotweezers as a tool to investigate protein conformational dynamics29. Ferritin is an iron carrier protein that exists in two states: apo-ferritin, which contains no iron, and holo-ferritin, which is filled with iron44,45. Ferrous iron enters the protein through 3-fold channels where it is oxidized to ferric iron and stored in the protein core46. Figure 6A depicts a typical trapping trace of apo-ferritin with a ferrous solution infused for over 20 min while the protein is trapped. The 20 s traces taken along the whole trace at points b-e provide insight into the changes occurring to the protein over time. In Figure 6B, apo-ferritin is trapped in a standard PBS buffer, and no significant changes are observed in the trace. Figure 6C, D show fluctuations in the S.D of the traces, which are caused by iron loading into the protein through its 3-fold channels, resulting in a more dynamic state (apo-) where the channels are open, and a more compact state (holo-) with the channels closed. Upon the ferritin molecule being filled with iron, it transitioned to its holoform, resulting in a stable trapping trace, as shown in Figure 6E. Probability density functions (PDF) in Figures 6B-E further showcase the changes the protein undergoes upon exposure to different solution conditions over time.
<img alt="Voltage vs. time graph showing iron loading process; experimental data filtered at 100 Hz, 1 kHz PDF." class="xfigimg" src="/files/ftp_upload/68093/68093fig06.jpg" data-altupdatedat="1788304762000" data-alt="Figure 6"> 
Figure 6: In situ iron loading into a trapped apoferritin. (A) Full transmission trace of a DNH with an apoferritin molecule trapped, followed by injecting a ferrous solution to the trapping site to observe ferritin's conformational changes associated with iron loading. (B) 20-s trapping trace of an apoferritin trapped before ferrous solution reached hotspot. (C, D) 20-s trapping traces after the apoferritin molecule was exposed to the ferrous solution. Blue and purple segments mark the higher and lower S.D of the trace, indicating flexible and rigid conformations of ferritin, respectively. (E) 20-s trapping trace after apoferritin was exposed to the ferrous solution for &gt;20 minutes. Probability density function (PDF) plots on the right show the distribution of transmission and are color-coded to the blue and purple segments. This figure has been modified from29. <a href="https://www.jove.com/files/ftp_upload/68093/68093fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Gold DNH sample mounted on the 3D printed flow cell. The sample is placed into a special slot and adhered to the flow cell using double-sided PET adhesive tape. Key parameters and associated measurements for our flow cell design are labeled. <a href="https://www.jove.com/files/ftp_upload/68093/SI-1%20Sample%20mounted%20in%20flow%20cell_amended.tif" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Back of flow cell with gold DNH sample mounted and inner wall labelled. The sample is sealed in the flow cell using duplicating silicone. <a href="https://www.jove.com/files/ftp_upload/68093/SI-2%20Inner%20wall%20of%20hole%20in%20flow%20cell.tif" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Diagram of the flow cell with gold DNH mounted with intake and outtake holes labeled. <a href="https://www.jove.com/files/ftp_upload/68093/SI-3%20Intake%20and%20outtake%20holes.tif" target="_blank">Please click here to download this File.</a>

Plasmonic nanotweezers use localized surface plasmon resonance in gold nanostructures to trap single nanoparticles, including proteins, within a nanometer-scale optical field. Changes in the scattered signal reveal protein presence and conformational dynamics, enabling monitoring without fluorophore modifications or surface tethering.

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes ...

monitoring-conformational-dynamics-single-unmodified-proteins-using

Research

JoVE Journal

Engineering

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5’ and 3’ ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A microchip fabrication process that incorporates plasmonic tweezers is presented here. The microchip enables the imaging of a trapped particle to measure maximal trapping forces.

Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders.

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in α-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

The present protocol describes a convenient approach to integrating optical trapping and surface-enhanced Raman spectroscopy (SERS) to manipulate plasmonic nanoparticles for sensitive molecular detection. Without aggregating agents, the trapping laser assembles plasmonic nanoparticles to enhance the SERS signals of target analytes for in situ spectroscopic measurements.

Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Learn plasmonic nanotweezers for real-time monitoring of label-free protein conformational dynamics at the single-molecule level. This method enables investigation of binding kinetics and free energy landscapes without fluorophore modifications.

Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers

Explore More Videos