Label-Free, All-Optical Detection, Imaging, and Tracking of a Single Protein

Arroyo, Jaime Ortega; Andrecka, Joanna; Spillane, Katelyn M.; Billington, Neil; Takagi, Yasuharu; Sellers, James R.; Kukura, Philipp

doi:10.1021/nl500234t

Cited by 153 publications

(170 citation statements)

References 28 publications

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“…In addition, iSCAT sensing can be used to visualize the motion of proteins (ref. 36) and monitor the association and dissociation kinetics of biomolecules and study their cooperative interactions 37 because it does not suffer from photobleaching. The sensitivity of iSCAT in our experiment was determined by the pixel well depth of the camera.…”

Section: Discussionmentioning

confidence: 99%

Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites

2014

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Detection of single analyte molecules without the use of any label would improve the sensitivity of current biosensors by orders of magnitude to the ultimate graininess of biological matter. Over two decades, scientists have succeeded in pushing the limits of optical detection to single molecules using fluorescence. However, restrictions in photophysics and labelling protocols make this technique less attractive for biosensing. Recently, mechanisms based on vibrational spectroscopy, photothermal detection, plasmonics and microcavities have been explored for fluorescence-free detection of single biomolecules. Here, we show that interferometric detection of scattering (iSCAT) can achieve this goal in a direct and label-free fashion. In particular, we demonstrate detection of cancer marker proteins in buffer solution and in the presence of other abundant proteins. Furthermore, we present super-resolution imaging of protein binding with nanometer localization precision. The ease of iSCAT instrumentation promises a breakthrough for label-free studies of interactions involving proteins and other small biomolecules. T here are more than 2,000 proteins secreted by the human body at concentrations that are below the detection limit of the current commercial biosensors 1 . To achieve a sufficient sensitivity for early and robust diagnosis of health hazards such as cancer and lethal infectious diseases, scientists have explored a fury of strategies based on mechanical 2 , electrochemical 3 and optical 4,5 interactions. The ultimate performance would be to count the analyte molecules one at a time. Furthermore, a practical biosensor would ideally detect the molecule of interest directly and without the need for labelling, amplification or sophisticated architectures.A well-established approach for label-free detection is via the vibrational signatures of molecules, for example via Raman spectroscopy. This method has an exquisite selectivity, but singlemolecule sensitivity has only been reached by enhancing the Raman cross-section in the near field of surfaces 6 or tips 7 . A common alternative strategy relies on the detection of the refractive index change due to analyte binding. The oldest and commercially available implementation of this technique records the shift of the plasmon resonance of a gold-coated prism 8 , whereby the specificity and selectivity are provided by surface chemistry 9 . While submonolayer detection is readily achievable in such a device, a single molecule does not manifest a significant change in the refractive index of the sensor medium. To increase the sensitivity, confined modes of plasmonic nanostructures [10][11][12] and dielectric microresonators 13,14 have been investigated, but these techniques are intrinsically limited. First, there is a compromise between the size of the sensor active area and its sensitivity. Higher sensitivity comes through confined intensity distributions and, thus, at the cost of more restricted active area, fewer binding receptors and thinner functionalization 15 . Se...

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Section: Discussionmentioning

confidence: 99%

Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites

2014

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“…This method has been demonstrated by Kukura et al [105] for the high spatial and temporal resolution detection and tracking of single viral particles. More recently, it has been successfully used in conjunction with suitable time average schemes and differential algorithms to image single actin filaments and single myosin 5a HMM molecules [101]. The uses of a coherent light source and of a sophisticated microscopy setup are probably the major limitations of the application of this technique outside of a laboratory.…”

Section: Digital Detection Of Bionanoparticlesmentioning

confidence: 99%

Emerging applications of label-free optical biosensors

et al. 2017

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Abstract:Innovative technical solutions to realize optical biosensors with improved performance are continuously proposed. Progress in material fabrication enables developing novel substrates with enhanced optical responses. At the same time, the increased spectrum of available biomolecular tools, ranging from highly specific receptors to engineered bioconjugated polymers, facilitates the preparation of sensing surfaces with controlled functionality. What remains often unclear is to which extent this continuous innovation provides effective breakthroughs for specific applications. In this review, we address this challenging question for the class of label-free optical biosensors, which can provide a direct signal upon molecular binding without using secondary probes. Label-free biosensors have become a consolidated approach for the characterization and screening of molecular interactions in research laboratories. However, in the last decade, several examples of other applications with high potential impact have been proposed. We review the recent advances in label-free optical biosensing technology by focusing on the potential competitive advantage provided in selected emerging applications, grouped on the basis of the target type. In particular, direct and real-time detection allows the development of simpler, compact, and rapid analytical methods for different kinds of targets, from proteins to DNA and viruses. The lack of secondary interactions facilitates the binding of small-molecule targets and minimizes the perturbation in single-molecule detection. Moreover, the intrinsic versatility of label-free sensing makes it an ideal platform to be integrated with biomolecular machinery with innovative functionality, as in case of the molecular tools provided by DNA nanotechnology.

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“…Interferometric scattering microscopy (iSCAT) is a label free platform capable of detecting, imaging and tracking the movement of single particles [155]. Optical detection of gold nanoparticles smaller than 10 nm in diameter using interferometric approaches was reported by Lindorfs and colleagues in 2004 [156].…”

Section: Interferometric Scattering Microscopymentioning

confidence: 99%

Detection and Digital Resolution Counting of Nanoparticles with Optical Resonators and Applications in Biosensing

Aguirre

Long

et al. 2018

Chemosensors

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Abstract:The interaction between nanoparticles and the electromagnetic fields associated with optical nanostructures enables sensing with single-nanoparticle limits of detection and digital resolution counting of captured nanoparticles through their intrinsic dielectric permittivity, absorption, and scattering. This paper will review the fundamental sensing methods, device structures, and detection instruments that have demonstrated the capability to observe the binding and interaction of nanoparticles at the single-unit level, where the nanoparticles are comprised of biomaterial (in the case of a virus or liposome), metal (plasmonic and magnetic nanomaterials), or inorganic dielectric material (such as TiO 2 or SiN). We classify sensing approaches based upon their ability to observe single-nanoparticle attachment/detachment events that occur in a specific location, versus approaches that are capable of generating images of nanoparticle attachment on a nanostructured surface. We describe applications that include study of biomolecular interactions, viral load monitoring, and enzyme-free detection of biomolecules in a test sample in the context of in vitro diagnostics.

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Label-Free, All-Optical Detection, Imaging, and Tracking of a Single Protein

Cited by 153 publications

References 28 publications

Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites

Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites

Emerging applications of label-free optical biosensors

Detection and Digital Resolution Counting of Nanoparticles with Optical Resonators and Applications in Biosensing

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