Drug Discovery Today � Volume 11, Numbers 13/14 � July 2006 REVIEWS

Downscaling functional bioassays bysingle-molecule techniquesFeng Hong and Douglas D. Root

University of North Texas, Department of Biological Sciences, Division of Biochemistry and Molecular Biology, PO Box 305220, Denton, TX 76203-5220, USA

In this short review we examine the potential of single-molecule assays in drug development and in basic

research to provide new types of information at the smallest assay scales. A key advantage of many

single-molecule assays is the requirement for conservative amounts of precious sample compared to

conventional assays. In addition, they measure processes that are not observed directly in molecular

ensembles. These advantages are balanced currently by difficulties in assay setup, preparation and

equipment expense. However, future developments will ameliorate these drawbacks with the

production of simpler, less expensive experimental systems for single-molecule assays.

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The advent of single-molecule assays opens new possibilities in

drug development and research. The downscaling of bioassays to

the single-molecule level has the potential to reduce the amounts

of sample required and increase the different types of information

that can be obtained. The difficulties in setting up and performing

single-molecule assays limit their implementation, but this might

improve as the technologies develop. In this review we introduce

some of the diverse single-molecule assays that are available and

discuss the practical aspects of their applications.

What is a single-molecule assay?In the most general sense, investigation of a system of interest at

the level of individual molecules is a single-molecule assay, which

can include detection, spectroscopy, manipulation, imaging and

computational simulation (Figure 1). Detection by optical meth-

ods yields positional information and quantification, often in real

time. Further spectroscopy of the sample can provide information

on structural changes either within a single molecule or relative to

another object, and can even be used to detect catalytic and

binding events. In addition, several experimental-manipulation

devices enable specific mechanical perturbation of the single

molecule. By contrast, high-resolution microscopy, such as elec-

tron microscopy of single molecules, can approach nanometer

resolution and traditionally requires immobilized samples,

although recent developments in atomic force microscopy

Corresponding author: Root, D.D. (droot@unt.edu)

640 www.drugdiscoverytoday.com 1359-6446/06/$ - s

(AFM) might allow real-time imaging in some cases. Although

theoretical in concept, computational simulations provide the

ultimate examination of the atomic resolution of a single mole-

cule. These areas offer new opportunities to test the effects of

drugs and characterize the molecular function of potential drug

targets.

Fluorescence microscopy is one of the most popular techniques

for single-molecule study, should adequate sensitivity be achieved.

Increasing the fluorescent signal from a single molecule by better

optical collection and higher excitation intensities is limited

primarily by photobleaching. Photobleaching can be reduced

substantially in vitro by the use of oxygen-scavenging systems

and reducing reagents. Another limitation is background, the

main sources of which are the fluorescence from residual impu-

rities, optical elements and light scattering. The following are

some basic geometries for increasing the signal:background-noise

ratio to detect single-molecule fluorescence:

(i) T

ee fron

otal internal reflection fluorescence microscopy (TIRFM).

TIRFM, which was developed by Dan Axelrod and colla-

borators [1], affords an important improvement in the

sensitivity of fluorescence microscopy. When high-angle

incident light is totally reflected from an interface between

two media of different refractive indices (e.g. glass and

water), the light (evanescent field) decays exponentially from

the interface and, thus, penetrates only 200 nm into the

optical medium with lower refraction number. This method

can be employed to selectively excite the sample that is only

t matter � 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.drudis.2006.05.003

Drug Discovery Today � Volume 11, Numbers 13/14 � July 2006 REVIEWS

FIGURE 1

Family tree of biological single-molecule assays. Typical single-molecule assays that involve visualization techniques can be described as belonging

to one of four major branches. Electron microscopy was the first technique to clearly visualize single macromolecules and it is an important validation

tool for other single-molecule studies. However, real-time functional applications of this technique are limited. Optical techniques are used mostwidely for functional single-molecule studies. Force microscopy and/or manipulation methods are relatively recent, but many diverse methods are

being developed. Computational single-molecule assays are becoming increasingly common as processing power and efficient algorithms make

them more accurate and practical. Although unlike experimental methods, computational techniques (such as molecular mechanics and dynamics) arepowerful visualization and analysis tools that can either assist other single-molecule assays or be used on their own. In this review, we focus on single-molecule

assays that are derived from visualization methods. Other single-molecule techniques that do not involve visualization, such as patch clamping are

reviewed in [60], which also describes optical trapping and magnetic tweezers.

Reviews�POSTSCREEN

located at the evanescent wave region. Thus, the background

outside of this region is greatly discriminated.

(ii) C

onfocal fluorescence microscopy (CFM). In CFM, the

excitation light is focused to a spot, producing a fluorescence

emission that is refocused to a detector pinhole, so that most

of the signals from other sources are excluded. Thus, the

background is greatly reduced and only the layer of specimen

that is in the confocal plane is imaged [2]. By changing the

focal positions, the whole specimen can be scanned to

reconstruct its 3D image. CFM is especially suitable for

investigating events in living cells and tissues in real time

and with high spatial resolution, but the pinhole limits the

amount of signal collected relative to wide-field microscopy.

(iii) N

ear-field scanning optical microscopy (NSOM). In NSOM, a

tapered optical fiber with subwavelength aperture size, often

coated with metal, serves as a near-field optical probe and is

scanned in close proximity to the sample [3]. A non-

propagating, oscillating, electric field extends several hun-

dred nanometers beyond the tip. The spatial resolution of

NSOM is limited by the size of the light source rather than by

the diffraction limit, and its optical resolution can be

<70 nm [3]. Only a few other fluorescence techniques, such

as stimulated emission depletion and structured illumina-

tion fluorescence microscopies, report comparable resolu-

tion [4]. A unique advantage of NSOM is that spectroscopic

and topographic information can be measured simulta-

neously. Disadvantages are low-power throughput, poor

reproducibility of the field distribution at the tip and the

possibility of either mechanical or electromagnetic pertur-

bation of the sample by the probe.

Often, fluorescence microscopy and spectroscopy are performed

simultaneously on single molecules, but other spectroscopic

single-molecule assays also exist. Another optical technique that

has been applied to single-molecule assays is surface enhanced

resonance Raman spectroscopy (SERRS). SERRS is a Raman

technique that provides greatly enhanced Raman signal from

Raman active molecules absorbed on certain surfaces. To obtain

single-molecule Raman spectra, individual molecules have to be

adsorbed on metallic nanoparticles, usually silver or gold, at a very

low concentration [5].

A different multifunctional tool for microscopy and spectro-

scopy is AFM. The basis of AFM performance is the repulsive and

attractive force between a nanometer-size tip (probe) and the

sample. A cantilever bends in response to forces between the tip

of the cantilever and the sample. This causes changes in the angle

of reflection of a probe laser beam from the back of the cantilever

and, thus, changes the position where the laser beam strikes the

detector. The sample is mounted on a piezoelectric stage, which

ensures high resolution in 3D positioning [6]. Biological samples

can be imaged in their native state, at a lateral resolution of 0.5–

1 nm and a vertical resolution of 0.1–0.2 nm, in real time and in

physiological conditions [7]. AFM can be more than an imaging

tool because single molecules can be manipulated by providing a

small (pN) force to the sample. A single biomolecule can be either

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REVIEWS Drug Discovery Today � Volume 11, Numbers 13/14 � July 2006

FIGURE 2

Practical limits of using small sample sizes. A key advantage of single-

molecule assays is the potential for downscaling to the ultimate level, butseveral practical limitations prevent the complete exploitation of this feature.

The extremely dilute concentrations that are required to achieve single-

molecule discrimination in these assays leads to large amounts of sample

loss, typically by non-specific adsorption to chambers, which increases theamount of sample needed for handling. Future developments might reduce

the amount of sample that can be handled.

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stretched or twisted by the AFM tip to study its mechanical

properties and structures [8]. Computational simulations of such

stretching can reproduce the force–distance curves that are

obtained experimentally, thereby adding atomic-resolution inter-

pretation to the measurements [9].

Even smaller forces than those of the AFM can be applied with an

optical trap. This is a highly focused laser beam that is used to trap

and remotely manipulate refractive particles [10]. A refractive par-

ticle near the focus will experience two types of force: a scattering

force, which is in the direction of light propagation; and a gradient

force, which is in the direction of the spatial light gradient. As the

result of a balance between the scattering force and gradient force,

the equilibrium position of the trapped particle is located slightly

down-beam from the focal point [11]. A small displacement

(<150 nm) from this position, results in a force imbalance that

tends to re-establish equilibrium, so the optical trap acts like a spring

and can be used as a force transducer in the pN range or lower.

Establishing precise position and force calibration is only practical

currently with spherical objects. Thus, microscopic beads are used

either alone or attached to objects of interest as handles to apply

calibrated force [12]. The current resolution is 0.1–0.4 nm and forces

as small as tens of fN have been measured [13,14].

Thediversityof single-molecule assays enables severalparameters

to be determined in a broad range of systems. By using these

techniques, the following properties can be measured in a single-

molecule assay: (i) localization of an individual molecule (even in a

living cell) [15,16], a site on an individual molecule [17], and

colocalization of more than one molecule [18]; (ii) fluorescence

intensity of dye molecules in living cells [15]; (iii) fluorescence

lifetime [19]; (iv) image and nano-structure of molecules [20,21];

(v) dynamics (reaction and conformation) [22–25]; (vi) intermole-

cular and intramolecular reactions [26,27]; (vii) distance between a

donor and an acceptor [28–30] or a donor and two acceptors [31];

(viii) orientation and rotation of molecules [32,33]; (ix) mechanical

properties of molecules [34–36]; and (x) folding and unfolding

intermediate states of macrobiomolecules [37]. Examples of experi-

mental systems that are currently studied at the single-molecule

level include, but are not limited to: (i) protein folding and unfold-

ing; (ii) enzyme catalysis; (iii) ion channels; (iv) signaling; (v) DNA,

RNA and their binding proteins; (vi) membrane structure;

(vii) molecular motors; and (viii) complex cellular structures. Thus,

there are many opportunities to exploit single-molecule assays.

How much sample is required for a single-moleculeassay?One attractive feature of using single-molecule assays for drug

development is the small size of the sample and valuable reagents,

which can enable a significant downscaling of conventional

assays. Several ml of a nanomolar concentration (or less) of sample

is sufficient for some single-molecule assays (Figure 2). However,

because single-molecule assays often have a low level of signal and

might have heterogeneous sample molecules, it usually takes more

repetitions of experiments (dozens to hundreds of repeats) than

ensemble experiments for satisfactory statistics and distribution

data for analysis. Also, the handling of small volumes of dilute

concentrations of sample can lead to large fractional losses of the

sample, especially from non-specific adsorption to vessel walls

during handling and storage. For small proteins and nucleic acids,

642 www.drugdiscoverytoday.com

the feasibility of chemically synthesizing single molecules in situ,

such as by modifying photolithographic techniques used on some

gene chips but with only a single molecule at each illuminated

spot, might reduce handling and storage problems. New oppor-

tunities exist for the development of technologies to deal with

such handling and storage problems (Figure 2).

In addition to analytes, other specialized materials, for exam-

ple, sensitive fluorescent dyes and modified surfaces, are needed

for a single-molecule assay. Fluorescent beads [38], dye-doped

nanoparticles [39] and quantum dots offer enhanced fluorescent

signals when larger probe sizes are tolerable [40,41]. Centroid

determinations on larger particles, such as microspheres, can

provide strong sensitivity even with brightfield microscopy

[42]. The chemical modification of molecules, particularly with

large probes, can sometimes require substantial quantities of

sample. Furthermore, dilute samples are often not suitable for

such processing, which provides a practical limit on the amount

of sample that is needed.

In experiments in which single molecules are immobilized on

surfaces, conjugating materials (e.g. BSA, biotin, streptavidin and

metal particles), and suitable surfaces (e.g. quartz cover slip) might

be required. Dilute samples typically adsorb slowly and ineffi-

ciently to surfaces, so that only a small fraction of the material

might be coated. In principal, if the sample quantity is highly

limited and not too fragile, the remaining sample can be recovered

and reapplied to a new area. Reducing the area over which the

sample is immobilized might be feasible for some single-molecule

assays. Microfluidics and spotting-array techniques reduce the area

over which a sample is applied to further improve sample utiliza-

tion [43]. The production of kits that facilitate single-molecule

assays has the potential to improve throughput, if sufficiently

reliable sources are available. AFM requires extremely flat, mod-

ified surfaces. Although these can be procured, they are usually

prepared fresh by the experimenter for optimum performance. The

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size and quality of the surface preparation will determine, in part,

the amount of sample that is required for a particular assay.

How fast are single-molecule assays?When planning a single-molecule assay, one must consider setup

and preparationtime,andthe timeneededtorecord and analyze the

data (Table 1). When planning high-throughput, single-molecule

assays, it is essential to identify steps that can be automated. Steps in

single-molecule assays differ widely in speed, depending on the type

of assayandthe breadth of information required.When implement-

inga newassay, setup is typically the rate-limiting step.However, for

computational single-molecule assays on large systems, data record-

ing is usually the slowest phase. Alternatively, if the data generated

are too complex to be analyzed easily and automatically, the ana-

lysis phase frequently becomes the longest part.

The setup of a single-molecule assay generally requires consid-

erable attention to both assay standardization and material pre-

paration. Assay standardization is most difficult during the initial

setup phase. Standardization is complicated by the observation

that different individual molecules seem to exhibit wide variations

in their responses. By contrast, ensemble measurements, by nat-

ure, contain a great deal of averaging that can reduce detection

variability. Consequently, more single-molecule observations rela-

tive to ensemble measurements are required to achieve a similar

level of confidence.

In many cases, the most problematic aspect of standardization is

determining the conditions under which a single molecule, rather

than an ensemble, is observed. Various arguments have been used

to support the singularity of the observed molecule including

sample dilution, frequency of response, photobleaching to zero

emission, anticipated magnitude of a signal, lowest unitary

response, lack of complexity in the data and microscopic visuali-

zation at high resolution [44]. Commonly, much time is spent

searching for a suitable single molecule to measure. The more

demanding the criterion for identifying single molecules, the

slower the assay. Often, not all observations need to be on a single

molecule; in such cases, occasional measurements of small groups,

in addition to single molecules, provide the desired information.

In some types of assays, calibration is a time-consuming com-

ponent of standardization. Optical-trapping devices, magnetic

traps and microneedle systems usually require extensive calibra-

tion, often for each measurement [45]. Calibration is somewhat

less demanding with atomic-force microscopes and other canti-

TABLE 1

Developments to increase throughput

Bottlenecks Possible developments

Setup Kit assays

Sample preparation Chemical synthesis

Observations Robotics

Analysis Automation

Computational speed Fast algorithms

Integration Inline coupling

Sensitivity Robust labels

Equipment availability Lower cost equipment

Validation Molecular imaging

lever-based systems, but is still quite demanding if the cantilever is

either changed or modified frequently. For higher-throughput

systems, designs and instruments should require minimal recali-

bration. Fluorescence measurements are usually the fastest to

standardize unless highly precise intensity comparisons between

widely differing microscopic fields are required.

Preparation of material is typically faster for single-molecule

assays than for bulk measurements because of the smaller sample

requirements. Chromatographic procedures of an analytical scale

are sufficient for many single-molecule assays, so intensive upscal-

ing of preparations can be avoided. Such analytical-scale prepara-

tions are also automated more easily, which can further reduce

time expenditures. However, if the sample must be analyzed by

slow procedures such as peptide mapping, as in some fluorescence

applications, then the time advantage is reduced or lost because of

difficulties in handling small sample amounts.

The time to record data can sometimes be faster for single-

molecule assays than for bulk measurements, such as in some force

measurements. Once a suitable molecule has been identified, the

recording of a mechanical measurement is not rate limiting and

occasionally is quicker than conventional force–transducer mea-

surements on larger samples. In AFM on a well-prepared sample,

hundreds of recordings can often be made in minutes. However,

many other single-molecule assays require longer acquisition times

that are either comparable to or exceed those of ensemble measure-

ments. The increased need for sensitivity might prolong exposure

times. If multiple types of measurements are to be performed on the

same single molecule, the manipulations require more care, and

repetitive measurements can be more crucial for single-molecule

assays.

Recording the data is not the rate-limiting step in most single-

molecule assays, but analysis of the data can be. The increased

simplicity of the single-molecule system can make the analysis per

measurement faster than that of comparable bulk samples. Con-

sequently, automation can greatly increase the speed the analysis

of single-molecule data. By contrast, more measurements and

increased noise in the data offset these speed advantages. In

particular, the sorting of the data to remove false positives, arti-

facts and contaminants becomes time-consuming if they either

occur frequently or are difficult to resolve from the intended data.

Why are single-molecule assays useful?A primary advantage of single-molecule assays is that they provide

unique information that cannot be obtained from ensemble mea-

surements (Figure 3). Consequently, comparing an ensemble mea-

surement with extrapolations from a single-molecule assay can

reveal complex interactions that result in differences between the

two. For instance, comparing recent studies of blebbistatin (which

inhibits myosin II) during in vitro motility assays of single actin

filaments sliding over myosin with its effects in live cells reveals

that blebbistatin produces high levels of free radicals when

exposed to blue light [46]. Also, the antibiotic microcin J25

increases pauses in transcription by bacterial RNA polymerase,

which is not resolved using ensemble assays [47].

The observation of single-molecule dynamics provides insight

into enzymatic mechanisms that are not obvious from ensemble

measurements [34]. Step sizes of motor proteins have been eval-

uated in greater detail by single-molecule assays [12,16,48]. The

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REVIEWS Drug Discovery Today � Volume 11, Numbers 13/14 � July 2006

FIGURE 3

New information from single-molecule assays. One of the major

advantages of downscaling assays to the single-molecule level is the ability to

extract types of information that is lost in measurements from ensembles.This new information might contribute to drug testing and target-system

characterization by, for example, enabling a more complete assessment of

the variability in responses and identifying transient structures that might

provide targets for drugs. Correlations between results from different types ofsingle-molecule assays are crucial to verify these essential new data.

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rotation of ATP synthase has been demonstrated in textbook

fashion [49]. Spontaneous unraveling of the myosin coiled coil

has been visualized in real time [50] and its flexibility measured in

multiple orientations [51]. Several nucleic acid binding proteins

including hexameric helicases, lambda exonuclease, topoisome-

rases and RNA polymerases have been shown to move along

nucleic acids with complex directional mechanisms [52–55].

The forces that are involved in unfolding proteins and nucleic

acids provide unique insights into the structure of these macro-

molecules and can be used to fingerprint various domains. Pro-

teins such as titin, myosin and b-amyloid each exhibit unique

force–distance curves that vary depending on the domain struc-

ture that is stretched [6,45,56]. Such quantification of the unfold-

ing and unbinding forces correlates with known biological

functions. Potentially, these molecular fingerprints can be used

to identify domain folds in otherwise uncharacterized structures.

Diversity in a molecular species is most evident when examined

at the single-molecule level. Variations in protein folding are often

detected less easily using ensemble averages. The variations can give

rise to a broad range in measurements of binding, catalysis, velocity,

metrology and flexibility, even among proteins with identical

sequences [57]. It is possible that drugs might be developed that

interact only with divergent states of the protein. Such states would

not be identified easily by determining average structures.

In some cases, simply observing and counting single molecules

with high precision might be an important task. Microscopic

fluorescence-autocorrelation methods facilitate counting, enzy-

matic analysis and diffusion characteristics of single molecules

in very small sample volumes that are not immobilized, such as

living cells [58]. Although the dispersion of single molecules across

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a surface is not determined readily by bulk measurements, it is

observed directly by single-molecule visualization. Substrate

release from single molecules can also be observed directly by

fluorescence, giving a measurement of kinetics. Unique photo-

physical properties, such as the blinking of green fluorescent

protein, have also been determined by single-molecule observa-

tion and this property might enable super-resolution imaging in

living cells by permitting saturated excitation at lower light inten-

sities [4].

In addition to providing new insights into the structure and

function of a molecule and its interaction with a drug, single-

molecule assays provide a great cost saving in some situations.

Most notably, if only small quantities of protein are available,

some types of single-molecule assays are the only feasible option.

For example, measuring the enzyme activity of some proteins

requires that they are expressed in mammalian cells in culture,

but the yield is far less than can be achieved in bacterial expression

systems. Many native proteins are only expressed in small quan-

tities, so their proteomic analysis would be enhanced greatly by

single-molecule analysis.

Molecular dynamics and mechanics simulations are performed

typically at the single-molecule level and offer great cost savings in

preliminary drug screening and the detailed atomic analysis of

structure. Large libraries of drugs can be screened for interaction

with a target atomic model at reasonable speeds, thereby eliminat-

ing the most unlikely candidates and reducing the costs of synth-

esis and animal testing. Although still less accurate than

experimental results, increasingly promising correlations between

macromolecular simulations and experimental results have been

identified for some types of simulations [59].

Single-molecule assays are not always cost-effective. In many

cases the equipment used is more expensive than for comparable

bulk measurements because more sensitive, delicate instruments

are needed. In addition, a higher level of operator expertise is often

required, which can lead to greater personnel expenditures, espe-

cially if there is an increase in the setup and processing time for the

given experimental design. Frequently, substantial changes in

setup and analysis are incurred when switching between assays

of different types of single molecules.

Concluding remarksDespite some drawbacks, single-molecule assays have several vir-

tues that make them either competitive with or superior to tradi-

tional assays for some applications. When sample quantities are

either limited or extremely expensive, single-molecule assays

usually have an advantage. If ensemble averaging prevents detec-

tion of the desired property, the single-molecule assay is the only

resort. Future increases in the diversity of single-molecule assays

will expand the types of properties that can be observed and the

speed and precision with which they can be analyzed, as well as

decrease the expense. The future will see rapid expansion of single-

molecule assays.

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