Exploring biomolecular interactions by single-molecule fluorescence
Introduction
Single-molecule detection has evolved from the limit of detection in chemical analysis into a modern method of systems investigation. The earliest works in the area of single-molecule detection were concerned primarily with the detection event itself [1]. In the past decade, there has been an increase in efforts to understand molecular systems at the stochastic level. Over the years, more researchers have demonstrated that simple instrumentation can achieve tremendous detection capabilities; a simple microscope, efficient filters and sensitive detectors are all that is required to detect single molecules. More importantly, more researchers are using those measurement capabilities to explore complex molecular systems.
Observing individual species – cells, atoms, or molecules – offers the advantage of detecting events obscured by the average, bulk signal. Such events include rare events, kinetically slow events, or heterogeneous populations that give mixed (combined) signals in bulk solution. A simple example of this is the observation of tandem conjugate dyes at the single-molecule and bulk (many-molecule) level. These tandem dyes are routinely used in single-laser, multi-color experiments to shift fluorophores to longer wavelengths via Förster resonance-energy transfer (FRET). A fluorescence spectrum of a tandem conjugate dye (e.g., the phycoerythrin (PE)-Alexa Fluor 647 (AF647) conjugate) typically reveals that the majority of the emission occurs from the AF647 dye (approximately 670 nm). A small percentage of the emission occurs at the PE emission maximum of 575 nm. At the bulk level, one could ask the question: “Do all PE-AF647 conjugates emit at both wavelengths, or are some of the PE-AF647 conjugates emitting at one of the wavelengths?” In the former case, inefficient energy transfer would be to blame; in the latter case, a fraction of the conjugates would be defective (i.e. complete failure to transfer energy to the second dye). This question is difficult to answer when measuring bulk concentrations. However, if one observes this system at the single-molecule level, individual molecules will emit at both wavelengths (incomplete energy transfer), the PE wavelength (faulty conjugate), or the AF 647 wavelength (complete energy transfer). This is a simple case, but it quickly illustrates that changing the measurement to a stochastic one can in many cases better elucidate the process under investigation.
Single-molecule detection implies that only one molecule is detected at a time. In truth, during the majority of the measurement, no molecules are detected. Rather, the appearance of a molecule in the system is marked by a photon burst of a certain length and intensity. To obtain sub-molecule measurements, the product of the concentration and the volume must be kept sufficiently low to ensure so-called double occupancies (>1 molecule in the probe volume) are minimized. In our laboratory, we typically operate in the 10–100-pM range. Since our probe volume is about 1 fL, there are typically 0.006–0.06 molecules in the probe volume. The rate of occupancy is dictated by Poisson statistics [2], and, at the upper end of our concentration range, the likelihood of detecting >1 molecule is 0.17%. The reduction in the probability of multiple occupancies is crucial to ensure that only individual events are being detected. There is also interest in using nanofabrication to decrease the measurement volume while maintaining a higher concentration, but proton-conduction effects in nano channels and other deviations from bulk-mass transport introduce many variables that are currently not well understood.
Analytical applications of single-molecule detection range from quantitative analysis to identifying individual species based on spectroscopic parameters. Extensive reviews have been written in recent years, and readers are directed there for a broad overview of single-molecule techniques [3], [4], [5]. This review focuses on the relatively new concept of observing molecular interactions by single-molecule spectroscopy. We have eschewed an exhaustive literature review and have instead focused on several key areas in the field where significant, transformative progress has been made recently.
Section snippets
Instrumentation
While numerous methods exist to detect single molecules, the two most common instrumental approaches to observing dynamic systems are confocal microscopy and total internal reflectance (TIR) microscopy [6], [7], [8]. Both methods isolate the sample to a small volume and allow for direct observation over time (Fig. 1). TIR microscopy is preferred in imaging applications as a two-dimensional image can be recorded simultaneously. However, confocal microscopy is more suitable for time-resolved
Protein-protein interactions
Single-molecule detection has emerged as a method to study protein-protein interactions that are difficult to investigate in the ensemble regime. Much can be learned about signal transduction and conformations of proteins from these studies. While many methods of single-molecule spectroscopy are available for protein-protein interactions, FRET and fluorescence-anisotropy studies dominate the methods employed to interpret these interactions.
Jäger and co-workers [20] exploited protein-protein
Future directions
Single-molecule detection has long been recognized as a powerful method for elucidating chemical and biochemical systems. Signals obscured by ensemble measurements have been shown to identify hidden mechanisms or rare events. Recent studies have largely shifted from the detection or identification of molecules in solution towards understanding biomolecular processes in situ. This advance in single-molecule detection is due in part to maturity in the instrumental approaches – both confocal and
Acknowledgements
The authors thank Horst Vogel and Matthew Paige for providing figures for this article. The authors also acknowledge a grant from the Welch Foundation, which supports many of the single-molecule investigations in their laboratory.
References (54)
- et al.
Anal. Chem.
(2005) - et al.
Anal. Chim. Acta
(2007) - et al.
FEBS Lett.
(2002) - et al.
Biophys. J.
(2004) - et al.
Methods
(2007) - et al.
Drug Discov. Today
(2006) - et al.
Anal. Biochem.
(2006) - et al.
Anal. Biochem.
(2002) Appl. Opt.
(1976)- et al.
Anal. Chem.
(1994)
Science (Washington, D. C.)
Anal. Chem.
Anal. Chem.
Science (Washington, D. C.)
Anal. Chem.
J. Microsc.
Anal. Chem.
Cytometry
Anal. Chem.
Proc. Natl. Acad. Sci. U. S .A.
Principles of Fluorescence Spectroscopy
Acc. Chem. Res.
J. Phys. Chem. A
J. Phys. Chem. A
Anal. Chem.
Protein Sci.
Nucleic Acids Res.
Cited by (17)
Nano-impact electrochemistry: Analysis of single bioentities
2020, TrAC - Trends in Analytical ChemistryCitation Excerpt :A central challenge in the field of analytical chemistry is to reduce the limit of detection (LOD) of analytes, e.g., molecules, nanoparticles or cells, to a single entity level, so that we can evaluate the heterogeneity and the intrinsically true properties of the analytes. A whole host of new techniques have been developed accordingly including scanning tunneling microscopy (STM) [1], fluorescence resonance energy transfer (FRET) [2,3], surface plasmon resonance microscopy (SPR) [4,5] and surface-enhanced Raman scattering (SERS) [6–8], all of which aim to achieve a detection sensitivity down to single entity level. However, these methods require highly experienced professionals, high analytical costs, and sophisticated handling procedures.
A fluorescence anisotropy method for measuring protein concentration in complex cell culture media
2014, Analytica Chimica ActaCitation Excerpt :Therefore, for the protein concentration measurements, we use another approach and exploit the intrinsic size (strictly speaking, the rotational correlation time) related information in the fluorescence emission. Fluorescence anisotropy characterizes the polarization state of the emitted light, and this in turn can be related to the physical size and/or restricted movement of a fluorophore [25,26]. Polarization measurements can introduce a new dimension to regular MDF spectroscopy of complex materials that offers a new insight on the origin of the emitting fluorophore [27,28].
Solving single biomolecules by advanced fret-based single-molecule fluorescence techniques
2013, Biophysical Reviews and LettersSuperwettable interface towards biodetection in confined space
2024, Nano ResearchAdvance in quantitative single-molecule detection
2017, Scientia Sinica Chimica