Nanoparticle‐Templated Molecular Recognition Platforms for Detection of Biological Analytes

Abraham G. Beyene1, Gozde S. Demirer1, Markita P. Landry1

1 California Institute for Quantitative Biosciences, QB3, University of California, Berkeley, Berkeley, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.10
Online Posting Date:  September, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Molecular recognition of biological analytes with optical nanosensors provides both spatial and temporal biochemical information. A recently developed sensing platform exploits near‐infrared fluorescent single‐wall carbon nanotubes combined with electrostatically pinned heteropolymers to yield a synthetic molecular recognition technique that is maximally transparent through biological matter. This molecular recognition technique is known as corona phase molecular recognition (CoPhMoRe). In CoPhMoRe, the specificity of a folded polymer toward an analyte does not arise from a pre‐existing polymer‐analyte chemical affinity. Rather, specificity is conferred through conformational changes undergone by a polymer that is pinned to the surface of a nanoparticle in the presence of an analyte and the subsequent modifications in fluorescence readout of the nanoparticles. The protocols in this article describe a novel single‐molecule microscopy tool (near‐infrared fluorescence and total internal reflection fluorescence [nIRF TIRF] hybrid microscope) to visualize the CoPhMoRe recognition process, enabling a better understanding of synthetic molecular recognition. We describe this requisite microscope for simultaneous single‐molecule visualization of optical molecular recognition and signal transduction. We elaborate on the general procedures for synthesizing and identifying single‐walled carbon nanotube‐based sensors that employ CoPhMoRe via two biologically relevant examples of single‐molecule recognition for the hormone estradiol and the neurotransmitter dopamine. © 2016 by John Wiley & Sons, Inc.

Keywords: fluorescence microscopy; molecular recognition; near‐infrared imaging; nanoparticles; neurotransmitter; nIRF TIRF hybrid microscope; single‐walled carbon nanotube (SWCNT); screening; single molecule imaging; sensors

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: A Microscopic Approach to Study Nanoparticle Molecular Recognition Coronae
  • Basic Protocol 2: Analyte Screening Using CoPhMoRe‐Based Optical Nanosensors: Testing Bulk Response of RITC‐PEG‐RITC‐SWCNT to Estradiol
  • Support Protocol 1: Synthesis of RITC‐PEG‐RITC‐SWCNT Estradiol Sensor
  • Support Protocol 2: Using nIRF TIRF to Interrogate CoPhMoRe in RITC‐PEG‐RITC‐SWCNT
  • Basic Protocol 3: Neurotransmitter Recognition Using Neurosensors
  • Support Protocol 3: Encapsulation of SWCNTS in (GT)15 DNA
  • Support Protocol 4: Encapsulate SWCNT in Polymers
  • Support Protocol 5: Using nIRF TIRF to Interrogate the (GT)15 DNA Dopamine‐Sensing Corona
  • Basic Protocol 4: Test Reversibility of SWCNT‐Based Molecular Sensors
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: A Microscopic Approach to Study Nanoparticle Molecular Recognition Coronae

  Materials
  • Supercontinuum (SC) light source (e.g., NKT EXR‐15)
  • Shutter (e.g., Thorlabs, cat. no. SH05)
  • Two 850‐nm long‐pass filters (e.g., Semrock, cat. no. FF875‐Di01‐25 × 36)
  • Neutral density filter set (e.g., Thorlabs, cat. no. FW2AND)
  • Plano‐convex lens (e.g., Thorlabs, cat. no. LA1256‐C)
  • Total internal reflection (TIR) lens (e.g., Thorlabs, cat. no. LA1380‐C)
  • Collimating lens
  • Long‐pass mirror (e.g., Thorlabs, cat. no. DMLP950)
  • Indium gallium arsenide (InGaAs) near‐infrared detector
  • Bandpass filter (various types, see below)
  • Electron multiplying charge‐coupled device (EM‐CCD) camera (e.g., Andor iXon EM‐CCD)
CAUTION: While working with the nIRF TIRF microscope, and particularly during its alignment and construction, always wear laser safety goggles and remove any reflective jewelry. The supercontinuum is a Class IV laser, and both direct and scattered beam exposure are hazardous to the eye and skin. To prevent eye exposure, be cognizant of the beam location and be aware of reflected beams. If the beam is not fully enclosed, a nominal hazard zone (NHZ) should be determined and an alarm, warning light, interlock safety system, and verbal countdown should be used during use or start‐up of the laser. During initial alignment, tune the laser output to no more than 10% power generation and use temporary neutral density filters along the beam path to minimize beam power. We suggest fully enclosing the laser path, once aligned, to reduce risk of laser‐related accidents.NOTE: Light path and image generation with nIRF TIRF (Fig.  B): Briefly, the microscope excitation and emission paths are constructed as follows: To excite SWCNTs and visible fluorophores over a broad optical range, the hybrid microscope excitation path uses a supercontinuum (SC) light source. The SC light source is a pseudo‐CW fiber laser technology, which emits across a wide range of wavelengths (400 nm to 2400 nm) by coupling a pulsed Ti:sapphire laser with a non‐linear photonic crystal fiber (Telford et al., ). The high‐coherence white light generated is then filtered (Chroma bandpass filters) to selectively produce 480‐nm to 800‐nm wavelength light for excitation. This range covers the requisite excitation maxima to visualize fluorophores and all excitation peaks of a typical SWCNT multi‐chirality sample (Fig.  A; Bachilo et al., ; Miyauchi et al., ; Salem et al., ). A shutter is used to allow illumination only during sampling. The SC light is conditioned through the following series of steps to image near‐infrared (nIR) and visible signals (refer to labels in Figure B).

Basic Protocol 2: Analyte Screening Using CoPhMoRe‐Based Optical Nanosensors: Testing Bulk Response of RITC‐PEG‐RITC‐SWCNT to Estradiol

  Materials
  • 50 mM PBS, pH 7.4
  • RITC‐PEG‐RITC‐SWCNT conjugate (rhodamine isothiocyanate‐PEG‐rhodamine isothiocyanate‐single‐walled carbon nanotube conjugate; synthesis provided in protocol 3)
  • 10 mM estradiol solution
  • 96‐well plate (Microtest 96 tissue culture plate, BD)
  • 1D OMA V near infrared InGaAs spectrometer (Princeton Instruments)

Support Protocol 1: Synthesis of RITC‐PEG‐RITC‐SWCNT Estradiol Sensor

  Materials
  • Amine‐difunctionalized polyethylene glycol (NH 2‐PEG‐NH 2, 5 kDa or 20 kDa; Creative PEGWorks or Nanocs)
  • Rhodamine isothiocyanate (RITC)
  • Dichloromethane (CH 2Cl 2)
  • Dimethylformamide (DMF)
  • N,N,‐Diisopropylethylamine (DIEA)
  • Diethyl ether (ether)
  • HiPco single‐walled carbon nanotubes (SWCNTs; NanoC or Unidym)
  • Sodium cholate (SoCh)
  • Stir bar and stir plate
  • 50‐ml Falcon tube
  • UV‐vis‐nIR absorption spectrometer (Shimadzu UV‐3101PC)
  • Sonicator (Cole‐Parmer)
  • SW32 Ti rotor (Beckman Coulter)
  • Centrifuge (Beckman Coulter)
  • 12‐ to 14‐kDa MWCO dialysis bag

Support Protocol 2: Using nIRF TIRF to Interrogate CoPhMoRe in RITC‐PEG‐RITC‐SWCNT

  Materials
  • Pierce BSA‐biotin (BSA‐Bt; Thermo Fisher Scientific)
  • NeutrAvidin (Thermo Fisher Scientific)
  • RITC‐PEG‐RITC‐SWCNT (see protocol 3) or any other conjugate of interest
  • 100 µM estradiol solution (see protocol 2) or analyte specific to the polymer‐SWCNT conjugate of interest
  • 535‐nm narrow‐bandpass filter (e.g., Semrock, cat. no. FF01‐535/50‐25)
  • 562‐nm dichroic mirror (e.g., Semrock, cat. no. FF562‐Di03‐25 × 36)
  • 585‐nm bandpass filter (e.g., Semrock, cat. no. FF01‐585/40‐25)
  • Pair of doublet lenses
  • iXon3 electron multiplying charge coupled device (EMCCD; Andor Technology)
  • Microfluidic chamber (e.g., Ibidi, sticky‐Slide VI 0.4)
  • 1.0 or 1.5 coverslip
  • Micropipet
  • Syringe

Basic Protocol 3: Neurotransmitter Recognition Using Neurosensors

  Materials
  • Polymer‐wrapped SWCNTs (see Support Protocols protocol 63 and protocol 74)
  • 10 mM PBS, pH 7.4
  • Neurotransmitters (Sigma‐Aldrich; see Figure  A)
  • 96‐well plate (Microtest 96 tissue culture plate, BD)
  • Micropipet
  • 1D OMA V near infrared InGaAs spectrometer

Support Protocol 3: Encapsulation of SWCNTS in (GT)15 DNA

  Materials
  • (GT) 15 DNA (Integrated DNA Technologies)
  • 0.1 M NaCl
  • HiPco SWCNTs (Unidym)
  • Sonicator (Cole‐Parmer)
  • Beckman Coulter Ultracentrifuge
  • UV‐vis‐nIR absorption spectrometer (Shimadzu UV‐3101PC)

Support Protocol 4: Encapsulate SWCNT in Polymers

  Additional Materials (also see protocol 5)
  • Sodium cholate (SoCh)
  • HiPco SWCNTs (Unidym)
  • Polymer of interest to conjugate to SWCNT
    • Single‐stranded DNA and RNA (N1 to N13; Integrated DNA Technologies)
    • Phospholipids (PL1 to PL12; Avanti Polar Lipids)
    • Phenoxy functionalized dextran (P1)
    • FITC‐PEG‐FITC (fluorescein isothiocyanate‐PEG‐fluorescein isothiocyanate; P2)
    • RITC‐PEG‐RITC (rhodamine isothiocyanate‐PEG‐rhodamine isothiocyanate; P3)
    • Boronic acid functionalized phenoxy‐PPEG8 (P4; Zhang et al., )
    • Folate‐poly (ethylene glycol)‐carboxylic acid (P5; Polyscitech)
  • Sonicator (Cole‐Parmer)
  • Beckmann Coulter Ultracentrifuge
  • UV‐vis‐nIR absorption spectrometer (Shimadzu UV‐3101PC)
  • 3.5 kDa molecular weight cutoff dialysis bag (Spectra/Por)

Support Protocol 5: Using nIRF TIRF to Interrogate the (GT)15 DNA Dopamine‐Sensing Corona

  Additional Materials (also see protocol 4)
  • (GT) 15‐Cy3‐single‐SWCNT (synthesis described in protocol 6 from either Cy3‐labeled (GT) 15 DNA [Integrated DNA Technologies] or (GT) 15 DNA [Integrated DNA Technologies] which was then labeled with Cy3 fluorophore)
  • 531‐nm narrow‐bandpass filter (e.g., Semrock, cat. no. FF01‐531/40‐25)
  • 562‐nm dichroic mirror (e.g., Semrock, cat. no. FF562‐Di03‐25 × 36)
  • 593‐nm bandpass filter (e.g., Semrock, cat. no. FF01‐593/40‐25)
  • Pair of doublet lenses

Basic Protocol 4: Test Reversibility of SWCNT‐Based Molecular Sensors

  Materials
  • (GT) 15‐Cy3‐SWCNT (synthesis described in protocol 6 from either Cy3‐labeled (GT) 15 DNA [Integrated DNA Technologies] or (GT) 15 DNA [Integrated DNA Technologies] which was then labeled with Cy3 fluorophore)
  • Pierce BSA‐biotin (Thermo Fisher Scientific)
  • NeutrAvidin (Thermo Fisher Scientific)
  • Microfluidic chamber (Ibidi, sticky‐Slide VI 0.4)
  • 1.0 or 1.5 coverslip
  • Micropipet
  • Syringe
  • Harvard Apparatus PHD 2000 syringe pump
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Adams, K.L., Puchades, M., and Ewing, A.G. 2008. In vitro electrochemistry of biological systems. Annu. Rev. Anal. Chem. (Palo. Alto. Calif.) 1:329‐355. doi: 10.1146/annurev.anchem.1.031207.113038.
  Bachilo, S.M., Strano, M.S., Kittrell, C., Hauge, R.H., Smalley, R.E., and Weisman, R.B. 2002. Structure‐assigned optical spectra of single‐walled carbon nanotubes. Science 298:2361‐2366. doi: 10.1126/science.1078727.
  Bisker, G., Ahn, J., Kruss, S., Ulissi, Z.W., Salem, D.P., and Strano, M.S. 2015. A mathematical formulation and solution of the CoPhMoRe inverse problem for helically wrapping polymer corona phases on cylindrical substrates. J. Phys. Chem. C 119:13876‐13886. doi: 10.1021/acs.jpcc.5b01705.
  Bisker, G., Dong, J., Park, H.D., Iverson, N.M., Ahn, J., Nelson, J.T., Landry, M.P., Kruss, S., and Strano, M.S. 2016. Protein‐targeted corona phase molecular recognition. Nat. Commun. 7. doi:10.1038/ncomms10241.
  Cho, E.J., Lee, J.‐W., and Ellington, A.D. 2009. Applications of aptamers as sensors. Annu. Rev. Anal. Chem. 2:241‐264. doi: 10.1146/annurev.anchem.1.031207.112851.
  Choi, J.H. and Strano, M.S. 2007. Solvatochromism in single‐walled carbon nanotubes. Appl. Phys. Lett. 90:223114. doi: 10.1063/1.2745228.
  Flavel, B.S., Moore, K.E., Pfohl, M., Kappes, M.M., and Hennrich, F. 2014. Separation of Single‐walled carbon nanotubes with a gel permeation chromatography system. ACS Nano 8:1817‐1826. doi: 10.1021/nn4062116.
  Giraldo, J.P., Landry, M.P., Kwak, S.Y., Jain, R.M., Wong, M.H., Iverson, N.M., Ben‐Naim, M., and Strano, M.S. 2015. A ratiometric sensor using single chirality near‐infrared fluorescent carbon nanotubes: Application to in vivo monitoring. Small 11:3973‐3984. doi: 10.1002/smll.201403276.
  Granite, M., Radulescu, A., and Cohen, Y. 2012. Small‐angle neutron scattering from aqueous dispersions of single‐walled carbon nanotubes with Pluronic F127 and poly (vinylpyrrolidone). Langmuir 28:11025‐11031. doi: 10.1021/la302307m.
  Hong, G., Diao, S., Antaris, A.L., and Dai, H. 2015. Carbon nanomaterials for biological imaging and nanomedicinal therapy. Chem. Rev. 115:10816‐10906. doi: 10.1021/acs.chemrev.5b00008.
  Jain, R.M., Tvrdy, K., Han, R., Ulissi, Z., and Strano, M.S. 2014. Quantitative theory of adsorptive separation for the electronic sorting of single‐walled carbon nanotubes. ACS Nano 8:3367‐3379. doi: 10.1021/nn4058402.
  Joo, C. and Ha, T. 2012. Single‐molecule FRET with total internal reflection microscopy. Cold. Spring. Harb. Protoc. 2012. doi: 10.1101/pdb.top072058.
  Kim, D., Koseoglu, S., Manning, B.M., Meyer, A.F., and Haynes, C.L. 2011. Electroanalytical eavesdropping on single cell communication. Anal. Chem. 83:7242‐7249. doi: 10.1021/ac200666c.
  Kruss, S., Landry, M.P., Vander Ende, E., Lima, B.M.A., Reuel, N.F., Zhang, J., Nelson, J., Mu, B., Hilmer, A., and Strano, M. 2014. Neurotransmitter detection using corona phase molecular recognition on fluorescent single‐walled carbon nanotube sensors. J. Am. Chem. Soc. 136:713‐724. doi: 10.1021/ja410433b.
  Landry, M.P., Kruss, S., Nelson, J.T., Bisker, G., Iverson, N.M., Reuel, N.F., and Strano, M.S. 2014. Experimental tools to study molecular recognition within the nanoparticle corona. Sensors 14:16196‐16211. doi: 10.3390/s140916196.
  Liu, Y., Dong, X., and Chen, P. 2012. Biological and chemical sensors based on graphene materials. Chem. Soc. Rev. 41:2283‐2307. doi: 10.1039/C1CS15270J.
  Liu, Z., Tabakman, S., Welsher, K., and Dai, H. 2009a. Carbon nanotubes in biology and medicine: In vitro and in vivo detection, imaging and drug delivery. Nano. Res. 2:85‐120. doi: 10.1007/s12274‐009‐9009‐8.
  Liu, Z., Tabakman, S.M., Chen, Z., and Dai, H. 2009b. Preparation of carbon nanotube bioconjugates for biomedical applications. Nat. Protoc. 4:1372‐1381. doi: 10.1038/nprot.2009.146.
  Marega, R., Aroulmoji, V., Bergamin, M., Feruglio, L., Dinon, F., Bianco, A., Murano, E., and Prato, M. 2010. Two‐dimensional diffusion‐ordered NMR spectroscopy as a tool for monitoring functionalized carbon nanotube purification and composition. ACS Nano 4:2051‐2058. doi: 10.1021/nn100257h.
  Miyauchi, Y., Chiashi, S., Murakami, Y., Hayashida, Y., and Maruyama, S. 2004. Fluorescence spectroscopy of single‐walled carbon nanotubes synthesized from alcohol. Chem. Phys. Lett. 387:198‐203. doi: 10.1016/j.cplett.2004.01.116.
  Moore, V.C., Strano, M.S., Haroz, E.H., Hauge, R.H., Smalley, R.E., Schmidt, J., and Talmon, Y. 2003. Individually suspended single‐walled carbon nanotubes in various surfactants. Nano Lett. 3:1379‐1382. doi: 10.1021/nl034524j.
  Oliveira, S.F., Bisker, G., Bakh, N.A., Gibbs, S.L., Landry, M.P., and Strano, M.S. 2015. Protein functionalized carbon nanomaterials for biomedical applications. Carbon 95:767‐779. doi: 10.1016/j.carbon.2015.08.076.
  Peluso, P., Wilson, D.S., Do, D., Tran, H., Venkatasubbaiah, M., Quincy, D., Heidecker, B., Poindexter, K., Tolani, N., and Phelan, M. 2003. Optimizing antibody immobilization strategies for the construction of protein microarrays. Anal. Biochem. 312:113‐124. doi: 10.1016/S0003‐2697(02)00442‐6.
  Perry, M., Li, Q., and Kennedy, R.T. 2009. Review of recent advances in analytical techniques for the determination of neurotransmitters. Anal. Chim. Acta 653:1‐22. doi: 10.1016/j.aca.2009.08.038.
  Rodriguez, P.C., Pereira, D.B., Borgkvist, A., Wong, M.Y., Barnard, C., Sonders, M.S., Zhang, H., Sames, D., and Sulzer, D. 2013. Fluorescent dopamine tracer resolves individual dopaminergic synapses and their activity in the brain. Proc. Natl. Acad. Sci. U.S.A. 110:870‐875. doi: 10.1073/pnas.1213569110.
  Roy, R., Hohng, S., and Ha, T. 2008. A practical guide to single‐molecule FRET. Nat. Methods 5:507‐516. doi: 10.1038/nmeth.1208.
  Saerens, D., Huang, L., Bonroy, K., and Muyldermans, S. 2008. Antibody fragments as probe in biosensor development. Sensors 8:4669‐4686. doi: 10.3390/s8084669.
  Salem, D.P., Landry, M.P., Bisker, G., Ahn, J., Kruss, S., and Strano, M.S. 2016. Chirality dependent corona phase molecular recognition of DNA‐wrapped carbon nanotubes. Carbon 97:147‐153. doi: 10.1016/j.carbon.2015.08.075.
  Shannahan, J.H., Brown, J.M., Chen, R., Ke, P.C., Lai, X., Mitra, S., and Witzmann, F.A. 2013. Comparison of nanotube‐protein corona composition in cell culture media. Small 9:2171‐2181. doi: 10.1002/smll.201202243.
  Skottrup, P.D., Nicolaisen, M., and Justesen, A.F. 2008. Towards on‐site pathogen detection using antibody‐based sensors. Biosens. Bioelectron. 24:339‐348. doi: 10.1016/j.bios.2008.06.045.
  Snow, E.S., Perkins, F.K., Houser, E.J., Badescu, S.C., and Reinecke, T.L. 2005. Chemical detection with a single‐walled carbon nanotube capacitor. Science 307:1942‐1945. doi: 10.1126/science.1109128.
  Telford, W.G., Subach, F.V., and Verkhusha, V.V. 2009. Supercontinuum white light lasers for flow cytometry. Cytometry Part A 75:450‐459. doi: 10.1002/cyto.a.20687.
  Zhang, J., Landry, M.P., Barone, P.W., Kim, J.‐H., Lin, S., Ulissi, Z.W., Lin, D., Mu, B., Boghossian, A.A., and Hilmer, A.J. 2013. Molecular recognition using corona phase complexes made of synthetic polymers adsorbed on carbon nanotubes. Nat. Nanotechnol. 8:959‐968. doi: 10.1038/nnano.2013.236.
  Zheng, M., Jagota, A., Strano, M.S., Santos, A.P., Barone, P., Chou, S.G., Diner, B.A., Dresselhaus, M.S., Mclean, R.S., and Onoa, G.B. 2003. Structure‐based carbon nanotube sorting by sequence‐dependent DNA assembly. Science 302:1545‐1548. doi: 10.1126/science.1091911.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library