Review

Cite This: Chem. Rev. 2019, 119, 293−321 pubs.acs.org/CR

Highly Sensitive and Multiplexed Protein Measurements

Limor Cohen†,‡,§ and David R. Walt*,†,‡
†
Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, United States
‡
Wyss Institute for Biologically Inspired Engineering and §Department of Chemical Biology, Harvard University, Boston,
Massachusetts 02115, United States

ABSTRACT: Proteins are involved in many biological processes. Misfolded, truncated,

or mutated proteins as well as over- or underexpressed proteins have been implicated in
many diseases. Therefore, detection and quantification of proteins is extremely
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important. Conventional techniques such as the enzyme-linked immunosorbent assay,

Western Blot, and mass spectrometry have enabled discovery and study of proteins in
biological samples. However, many important proteins are present at low
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concentrations, rendering them undetectable using conventional techniques.

Furthermore, limited ability to simultaneously measure multiple proteins in a sample
has constrained our ability to fully study the proteome. In this review, we
comprehensively discuss approaches for protein detection. We first discuss the
fundamentals of proteins and protein assays, including affinity reagents, surface functionalization, assay formats, signal detection,
and multiplexing. We then discuss the challenges with these methods and review existing methods for highly sensitive and
multiplexed protein detection. Finally, we review recent advances in protein detection from the literature and discuss challenges
and future directions.

CONTENTS 6.1.1. Meso Scale Discovery (MSD) 305

6.1.2. Single-Molecule Arrays (Simoa) 306
1. Introduction 294 6.1.3. Single-Molecule Counting (SMC) 306
2. Proteins 294 6.1.4. Single-Cell Western Blot 307
3. Fundamentals of Protein Assays 295 6.2. Highly Multiplexed Methods for Protein
3.1. Affinity Reagents 295 Detection 307
3.1.1. Antibodies 295 6.2.1. SOMAscan Assay 307
3.1.2. Aptamers 296 6.2.2. Luminex 307
3.1.3. Other Affinity Reagents 296 6.2.3. Mass Cytometry (CyTOF) 309
3.2. Surface Functionalization 296 7. Advances from the Literature 309
3.3. Assay Formats 298 7.1. Advances in Labeling and Signal Detection 309
3.4. Signal Detection 298 7.1.1. Upconverting Nanoparticles 309
3.5. Multiplexing 299 7.1.2. Photoelectrochemical Detection (PEC) 309
4. Current Commonly Used Methods for Protein 7.1.3. Optical Ring Resonators 310
Detection 299 7.1.4. Surface-Enhanced Raman Scattering
4.1. Detection of Total Protein 299 (SERS) Tags 310
4.2. Enzyme-Linked Immunosorbent Assay 7.2. Miniaturization 310
(ELISA) 300 7.2.1. Microcantilever-Based Assays 310
4.3. Enzyme-Linked Immunospot (ELISPOT) 300 7.2.2. Ultrasmall Containers 311
4.4. Western Blot 301 8. Conclusion and Future Directions 313
4.5. Protein Microarrays 301 Author Information 313
4.6. Flow Cytometry 301 Corresponding Author 313
4.7. Proximity Ligation Assay (PLA) and other ORCID 313
Nucleic Acid-Based Detection Methods 302 Notes 313
4.8. Mass Spectrometry 302 Biographies 313
4.9. Lateral Flow Assay (LFA) 303 References 314
4.10. Surface Plasmon Resonance (SPR) 303
4.11. Optical Imaging 304
5. Challenges with Protein Detection 304
6. Emerging Methods for Ultrasensitive and Highly
Special Issue: Chemical Sensors
Multiplexed Measurements 305
6.1. Ultrasensitive Methods for Protein Detec- Received: April 20, 2018
tion 305 Published: August 28, 2018

© 2018 American Chemical Society 293 DOI: 10.1021/acs.chemrev.8b00257

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1. INTRODUCTION ubiquination, deamidation, oxidation, sulfation, and nitration.

The five major classes of covalent modifications are shown in
Proteins are macromolecules involved in important biological
Figure 1.4 In some cases, detection of a specific post-
functions including replicating genomic information, regulating
transcription, signaling, providing structure, catalyzing reac-
tions, and transporting molecules. Many proteins are
dysregulated and differentially expressed in disease and can
serve as drug targets or biomarkers. Therefore, tools for
measuring and detecting proteins are essential for fully
characterizing these functions.
Several challenges exist with protein detection methods.
First, a sample may contain many interfering molecules that
make it difficult to detect a specific protein. Second, many
proteins may be present at low concentrations in a biological
sample. Third, concentrations of different proteins can vary by
many orders of magnitude. Finally, protein processing, such as
post-translational modifications and spliced variants that result
in different isoforms, can make it difficult or complicated to
detect the specific protein of interest. Common techniques
currently employed for protein detection include the Western
blot, mass spectrometry, and the enzyme-linked immunosorb-
ent assay (ELISA). These techniques have provided a wealth of
information on proteins and their function; however, they may
suffer from shortcomings in throughput, multiplexing capa-
bilities, specificity, and sensitivity. In the past few years, many
tools have been developed to overcome these challenges.
In this review, we comprehensively discuss existing and
innovative analytical tools for protein detection and
quantification. First, we briefly discuss the fundamental
properties of proteins. We then discuss fundamentals of
protein assays, including affinity reagents, surface functional-
ization, assay formats, signal detection, and multiplexing. We
then review commonly used methods for protein detection. Figure 1. Five major types of covalent modifications to protein side
We then discuss the challenges with these methods and review chains: phosphorylation, acylation, alkylation, glycosylation, and
oxidation. Reprinted with permission from ref 4. Copyright 2005
existing methods for highly sensitive and multiplexed protein John Wiley and Sons.
detection. Finally, we review advances in protein detection
techniques from the literature and discuss challenges and translational modification on a protein may be desirable over
future directions. detection of the unmodified protein because it may be the
functional or disease-causing species. Some protein detection
2. PROTEINS methods first start by treating the protein with a glycosylase,
Proteins are a class of biological macromolecules that play which removes glycans. This treatment is designed to reduce
important roles in many biological processes. Proteins are the complexity of the sample but may remove important PTMs
essential for replicating genomic information, regulating gene that are key to understanding the sample being measured.
expression, transcribing mRNA, signaling, catalyzing metabolic These modifications play critical roles in the function of
reactions, and transporting molecules. Disrupted protein proteins and can be dysregulated in disease.5−9 For example,
expression as well as dysfunctional mutated or misfolded approximately 30% of proteins encoded by the human genome
proteins are associated with multiple diseases, such as are phosphorylated, with irregular phosphorylation patterns
Alzheimer’s disease and sickle cell anemia. Thus, detecting associated with many human diseases.10 Thus, it is not only
specific proteins is important. Several features of proteins are important to detect specific proteins but also to detect specific
important for their detection and are discussed in this section. modifications on certain proteins. Additionally, proteins range
First, the proteome consists of tens of thousands of proteins, in size from very small monomeric proteins to large
and thus, detecting a specific protein in a complex matrix is macromolecular complexes, in which proteins interact with
challenging. The Human Genome Project revealed that other molecules such as RNA, DNA, or other proteins.
different proteins can be encoded by the same genes. It has Therefore, it is often challenging to detect a specific protein or
been estimated that the human genome contains approx- a specific modification on a protein in a complex biological
imately 20 000 protein coding genes,1 yet the number of sample.
proteins is considerably higher as a result of variations in DNA Second, some proteins of interest may be present at low
sequence, alternative mRNA splicing, post-translational mod- levels, limiting the ability to detect them. It can be particularly
ifications, and proteolytic cleavage.2 A mammalian cell contains difficult to measure low levels of a given protein in a complex
a total of 109 protein molecules, and a bacterial cell contains a biological sample that contains high levels of other potentially
total of 106 protein molecules.3 Proteins are modified by interfering molecules. Third, it is often necessary to detect
covalent post-translational modifications including phosphor- more than one protein in a single sample. Many proteins work
ylation, acetylation, methylation, acylation, glycosylation, in networks to exert their function. Some proteins are
294 DOI: 10.1021/acs.chemrev.8b00257
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overexpressed, while others are under expressed. Protein 3.1.1. Antibodies. Antibodies, major components of the
measurements on the omics scale are highly advantageous vertebrate immune system, bind to foreign molecules known as
and provide unbiased detection of proteins. Measurements of antigens. Most antigens are proteins. An antibody binds
many proteins simultaneously is challenging because proteins specifically to a structure on the antigen called an epitope.
in a biological sample can differ in concentration by several Several classes of antibodies exist, with Immunoglobulin G
orders of magnitude.11 Additionally, levels of a target protein (IgG) being a major class and most commonly used in
can vary substantially between different biological samples, bioanalytical applications. IgGs have four polypeptide chains
which further complicates detection. Finally, the particular consisting of two heavy chains and two light chains that form a
structural form of a protein may depend on its native cellular 150 kDa molecular complex (Figure 2). The variable domains
context. To analyze proteins, it is often necessary to alter their
native environment or remove them from it completely. For
example, hydrophobic membrane proteins that are embedded
in the lipid bilayer may not be stable in aqueous environments.
Therefore, when removing a protein from its native environ-
ment for analysis, it is important to determine if such
manipulations will affect the analysis.

3. FUNDAMENTALS OF PROTEIN ASSAYS

Methods to analyze proteins can be broadly classified into
three categories. The first category is protein detection using
solution-based assays, which will be the focus of this review.
The second category is protein detection using mass
spectrometry-based methods. The third category is structural Figure 2. IgG structure showing two different molecular depictions
analysis of proteins using methods such as X-ray crystallog- on the left and right sides. IgGs consist of two heavy chains and two
raphy, NMR, Cryo-EM, and circular dichroism (CD). The light chains that are linked by a disulfide bond. IgG contains two
antigen-binding sites. Modified and reprinted with permission from
latter two categories have been thoroughly reviewed elsewhere ref 20. Copyright 2017 Annual Reviews.
and will not be the focus of this review.12,13
A protein assay typically consists of three major of the light and heavy chains associate to form the antigen-
components: an affinity reagent, a signal transducer, and a binding domain, also known as the Fab. In 1993, it was
detector.14 The role of the affinity reagent is to specifically bind
discovered that sharks16 and camelids, such as camels and
to the target protein molecule. This binding enables significant
purification of the protein from the complex sample being llamas,17 produce antibodies that do not have a light chain.
analyzed. The signal transducer then converts the binding These single-domain antibodies are known as Nanobodies or
event into a measurable signal, such as an optical, electro- VHHs (Figure 3).18 Single-domain antibodies have lower
chemical, or mechanical signal. The detector, such as a charge- molecular weights (90 kDa) than traditional antibodies (150
coupled device (CCD), then converts the signal into a digital kDa) and therefore may be better affinity reagents.19
readout. In this section we discuss affinity reagents, surface Antibodies are generally produced by affinity maturation in a
functionalization, assay formats, signal detection, and multi- living organism. Antibody diversity is attained by combinatorial
plexing. biosynthesis based on genetic recombination capable of
3.1. Affinity Reagents
An affinity reagent is a molecule that recognizes a specific
protein. Binding of the protein to the affinity reagent is
converted into a measurable signal. Affinity reagents may need
to be adsorbed or covalently attached to a surface. Approaches
for covalent conjugation of biomolecules have been thoroughly
described elsewehere.15 Additionally, affinity reagents need to
interact with the signal transducer either directly or via a
secondary molecule that can interact with the signal trans-
ducer. These interactions must not affect the performance of
the measurement by sterically hindering binding to the protein
or affecting the performance of the signal transducer.
There are several important characteristics of protein affinity
reagents including high affinity (low dissociation constants),
high specificity (ability to recognize the target protein in a
sample containing many other potentially interfering mole-
cules), cost, stability, and reproducibility. The most commonly Figure 3. Single-domain antibody structure. Single-domain antibodies
are much smaller compared to IgGs. Single-domain antibodies do not
used affinity reagents for proteins are antibodies and aptamers. have a light chain and are derived only from the heavy chain. Single-
Other affinity reagents such as molecularly imprinted domain antibodies are naturally found in camelids and sharks. (v,
polymers, lectins, peptides, and antibody mimetics can also variable; HCAb, heavy-chain-only antibodies; H, heavy chain).
be used. These affinity reagents will be discussed in this Modified and reprinted with permission from ref 21. Copyright
section. 2014 Nature Publishing Group.

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Chemical Reviews
producing more than 108 different antibodies that have
different binding specificities.22 The most common antibody
preparations are polyclonal and monoclonal antibodies.
Polyclonal antibodies are generated in vivo via affinity
maturation. Therefore, polyclonal antibodies recognize differ-
ent epitopes of a given antigen. Monoclonal antibodies are
derived from a hybridoma, a fusion of a B cell with an immortal
cell line, and were first described in 1975.23 Since monoclonal
antibodies are derived from a clone of identical B cells, they
recognize a single epitope.
In some cases, it is preferable to use one clone over another;
for example, when detection of a specific post-translational
modification is required, a monoclonal preparation is necessary
since a polyclonal preparation will detect all of the epitopes on
the protein. On the other hand, in some cases a polyclonal
preparation is favorable−for instance, when detecting the
presence of all proteins containing a particular epitope,
regardless of the PTM. A disadvantage of polyclonal antibodies
is the potential lack of reproducibility between different
reagent lots. Once a polyclonal preparation is depleted an
animal must be immunized again to produce more polyclonal
antibodies. The second polyclonal batch will likely have a
different composition and may perform differently than the
first batch, requiring new assay conditions to be determined
and new calibrations to be performed.
Several approaches for in vitro antibody production have
been developed. These include phage, yeast,24 and ribosome
display,25,26 which have been reviewed elsewhere.24,27,28 These
approaches are advantageous for high-throughput antibody
production as well as production of antibodies against toxic
molecules and molecules that do not elicit an immune
response in an animal. Another advantage is the ability to
incorporate a tag, such as a his-tag, for antibody immobiliza-
tion and conjugation to various labels. Finally, these
recombinant preparations may reduce the cost of antibody
production.
Antibody−antigen interactions are particularly strong and
have low KD values. In vivo affinity maturation, with repeated
exposure of an animal to the same antigen, typically results in
antibodies with affinities in the nanomolar to picomolar
range,29 but directed evolution of antibodies with affinities in
the femtomolar range have been reported.30 Antibodies are
generally highly specific to their target, although nonspecific
binding can occur. Overall, due to their high affinity and
specificity, antibodies are key reagents for protein detection.31
Antibodies can be easily integrated into protein assays.
Antibodies have multiple reactive side chains that can serve as
attachment sites for conjugation to solid supports, such as
magnetic beads or nanoparticles, or labels that act as signal
transducers. Antibodies are usually covalently attached to
surfaces or detection labels. Another approach is to utilize
biotinylated antibodies that can interact with streptavidin,
which has been attached to a surface or a signal transducer.
However, covalent conjugation may affect interaction with the
target protein. Modifying a molecule that can interact with the
antibody may overcome this challenge. A secondary antibody,
Protein A, or Protein G, which bind to the Fc region of the
antibody, may be used.
3.1.2. Aptamers. Aptamers are single-stranded DNA or
RNA oligonucleotides that can bind to proteins with high
affinity and specificity.32,33 First described in 1990,34,35
aptamers are created by an in vitro process known as
SELEX, systemic evolution of ligands by exponential enrich-
296
Review
ment. The SELEX process starts with a large combinatorial
library of DNA or RNA oligonucleotides of random sequences.
These sequences are added to the target protein, usually
attached to a solid support, and some bind with high affinity.
The sequences that do not bind to the target protein are then
separated from those that bind to the target protein. The
bound sequences are eluted from the protein, amplified, and
undergo another round of selection. This process is repeated,
typically between 8 and 15 rounds, with each iteration
subjecting the bound sequences to higher stringency to elicit
stronger binders. The targets are then cloned and sequenced.
Aptamers have several advantages for use as affinity reagents
such as low cost, high stability, and high specificity, with
dissociation constants in the femtomolar to picomolar
range.36−38 Once an optimal sequence has been determined,
they can be easily and reproducibly generated by chemical
synthesis. Additionally, DNA aptamers are highly stable
reagents such that bound proteins can be removed either
chemically or thermally to regenerate the free aptamer for
another round of binding. Another advantage of aptamers is
that they can be generated against almost any protein, even
toxic proteins and those that are not immunogenic. Aptamers
can be easily integrated into many protein assay formats by
substituting for antibodies and are easily labeled and modified
with various molecules and functional groups. Aptamers are
highly stable and can also be regenerated for numerous uses.
The SOMAscan assay, which is described in section 6.1.1, uses
aptamers as affinity reagents.
3.1.3. Other Affinity Reagents. Other than antibodies
and aptamers, additional affinity reagents are available
including molecularly imprinted polymers (MIPs),39,40 protein
ligands, lectins, and antibody mimetics. The protein itself or
other peptide ligands can be used to detect protein−protein
interactions. For example, when one needs to detect antibodies
to determine if an immune response has occurred, the protein
can be used to bind to the target antibodies. Lectins, such as
wheat germ agglutinin or concanavalin A,41 bind to glycans on
glycoproteins and are commonly used. Alternative molecules
that mimic antibodies, known as antibody mimetics, are
proteins that are structurally unrelated to antibodies and can
bind to targets specifically through protein-engineering
approaches.20
3.2. Surface Functionalization
In many protein assays, the target protein must first come into
contact with an affinity reagent that is immobilized on the
surface. Therefore, the performance of a protein assay is highly
dependent on the chemical modification of the surface with an
affinity reagent.42 Approaches to attach an affinity reagent onto
the surface include noncovalent and covalent immobilization.
Strategies for covalent immobilization chemistries have been
thoroughly described.15 Noncovalent surface attachment can
be achieved by direct electrostatic interactions between the
surface and the affinity reagent. Alternatively, secondary
molecules such as biotin−streptavidin or attachment via
interaction of Proteins A and G to the Fc region of the
antibody can also be used. Self-assembled monolayers (SAMs)
are also commonly used to attach affinity reagents to a
surface.43 Finally, encapsulation of the affinity reagent is
another approach. All of these strategies can be used to attach
an affinity reagent to the surface (Figure 4).44
There are two different implementations for using surface-
bound affinity reagents. The first implementation is one in
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which the target protein must diffuse to the surface, for

Figure 4. Five strategies used for bioconjugation of affinity reagents to
surfaces via (i) electrostatic interactions, (ii) direct interaction with
the surface, (iii) secondary interactions, (iv) covalent immobilization,
and (v) encapsulation. Reprinted with permission from ref 45.
Copyright 2013 American Chemical Society.
example, when using an affinity reagent immobilized onto the
surface of a traditional microtiter plate. The second
implementation is when the affinity reagent is immobilized
onto the surface of micro- or nanoparticles and the target
protein and the surface are both in solution. Solution-based
assays enhance and accelerate the binding kinetics and capture
efficiency of the target protein due to superior mass transport
of the protein to the surface. Another advantage is the higher
surface to volume ratio of micro- and nanoparticles, which
further improves the capture efficiency. Another consideration
is the orientation of the affinity reagent on the surface and the
surface density or number of affinity reagents per area. Both
have been shown to affect the binding efficacy of the target
protein.46,47 Recently, several studies have assessed the
relationship between capture efficiency and antibody function-
alization of gold nanoparticles.45,48−51 Due to their favorable
properties, nanomaterials are becoming more popular for use
in protein detection techniques. These nanomaterials include
carbon nanotubes, polymer nanowires, and zinc oxide
nanorods.52−55
Reduction of nonspecific binding to the surface is also
important. Methods for passivating the surface include using
BSA56 or various neutral or hydrophilic macromolecules such
as poly(hydroxyethyl methacrylate), poly(acrylamide), and
poly(ethylene glycol).57−60
Finally, another consideration is the ability to regenerate the
binding capabilities of the surface for multiple independent
measurements of proteins.61 Regeneration is achieved by
overcoming the attractive forces between the affinity reagent
and the bound protein, ideally without destroying the binding
capabilities of the former. Strategies to regenerate the binding

Figure 5. Immunoassay formats. (a) Label-free immunoassay. Binding of the target protein to capture antibodies immobilized onto a surface results
in a measurable signal. (b) Sandwich immunoassay in which the detection antibody is labeled directly with a tag such as a fluorophore. (c)
Sandwich immunoassay in which an enzyme is used to amplify the signal. (d) Competitive immunoassay in which the target protein competes with
the labeled protein from binding to the capture reagent. (a−d) Heterogeneous immunoassays in which the capture reagent is immobilized onto a
surface and the unbound sample components are washed away. (e) Homogeneous immunoassay in which the reagents are in solution and are not
separated from other unbound sample components.

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Chemical Reviews
capabilities of surfaces have been developed;62 however, it is
still quite challenging to regenerate the surface because the
performance is compromised due to reduced binding affinity,
increased nonspecific binding, and thus lower sensitivity. Some
affinity reagents, such as aptamers, may be more amenable to
regeneration than antibodies. The main benefits of surface
regeneration are reduced cost and the ability to monitor
changes in concentration over time without changing the
affinity capture surface.
3.3. Assay Formats
The two most common formats for protein assays are
noncompetitive and competitive assays. Noncompetitive assays
can be further broken down into label-free assays (Figure 5a)
and sandwich assays (Figure 5a−c). Both label-free and
sandwich assays require a capture reagent that specifically
binds to the target protein. In a label-free assay, binding of the
target protein to the capture reagent results in a measurable
change. In a sandwich assay, a second affinity reagent binds to
a different epitope on the target protein, forming a “sandwich.”
The second affinity reagent is labeled with a tag, such as a
fluorophore (Figure 5b), or an enzyme (Figure 5c) that
produces molecules that can be detected. In a competitive
assay (Figure 5d), the surface is coated with a capture reagent
that binds to the target protein. A sample containing the target
protein and a known amount of labeled target protein are
added. The target protein competes with the labeled protein
from binding to the capture reagent. When low levels of the
target proteins are present relative to the labeled protein, the
labeled protein can bind to the capture reagents to produce a
signal. With increasing levels of the target protein, binding of
the labeled protein to the capture reagent decreases. Therefore,
the concentration of the target protein is inversely proportional
to the signal. A competitive assay for protein detection is less
commonly used than the noncompetitive assay. However, it is
often used to detect antibodies or when only one affinity
reagent is available.
Protein assays can be homogeneous (Figure 5e) or
heterogeneous (Figures 5a−d). In a homogeneous assay, the
reagents are in solution and do not require separation from
other unbound sample components, while in a heterogeneous
assay, the capture reagent is immobilized onto a surface and
the unbound sample components are washed away. Hetero-
geneous assays are more commonly used than homogeneous
assays.
3.4. Signal Detection
Interaction of a protein with an affinity reagent must be
converted into a measurable signal. Signal transduction
approaches fall into two general categories: chemical and
physical (or label-free) transduction. Chemical transduction is
a change in chemical composition coupled to a binding event,
for example, when an enzyme catalyzes the formation of a
fluorescent product. Physical transduction is a measurable
change in the physical properties in response to the interaction
of the affinity reagent with the protein. Because physical
transduction methods do not require a chemical label, they are
also known as label-free transduction. Several reviews have
been written that provide more detail on optical label-free
detection methods including theoretical discusions.63−67
Chemical and label-free signal transduction approaches
relevant to protein detection include optical, electrochemical,
and mechanical signal transduction.
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Review
Optical detection is most commonly used for signal
transduction. Optical detection is a signal transduction method
based on measuring changes in the amount of light absorbed
or emitted at a specific wavelength, typically in the ultraviolet
(UV) or infrared (IR) regions. The affinity reagent is labeled
with a molecule that can be optically detected. Since the ability
to detect the protein analyte depends on detecting the optical
label, the label needs to either have a high extinction coefficient
for absorbance measurements or have a high quantum yield for
fluorescence emission measurements. In addition, there should
be a sufficient degree of labeling on the affinity reagent.
Fluorescent labels are more commonly used in optical
detection. Two key properties of fluorophores are their Stokes
shift and quantum yield. A larger Stokes shift results in better
signal separation from the excitation wavelength. The quantum
yield, along with the molar extinction coefficient, determines
the overall brightness of the fluorophore.68 Numerous
fluorescent dyes and their derivatives have been developed to
increase the brightness and quantum yield.69 The most
common fluorescent core structures are water-soluble con-
jugated organic molecules that are derivatives of cyanine,
fluorescein, rhodamine, and coumarin. Derivatives with
aromatic ring constituents, such as electron-donating or
-withdrawing groups, can have drastic effects on fluorescence
and quantum yield. Fluorescent molecules other than small
organic dyes can also be used for optical detection. These
include naturally fluorescent protein molecules, such as
phycoerythrin and green fluorescent protein (GFP). However,
these molecules are larger than organic dyes and may sterically
hinder the protein from binding to the affinity reagent.
There are several limitations to detecting a fluorophore that
has been directly conjugated to an affinity reagent. First, the
protein-binding site on the affinity reagent may be unavailable
for binding to the protein. Second, when multiple fluorophores
are proximal to each other, quenching could occur. Finally, a
limited number of fluorophores can be attached to an affinity
reagent either because more labels will affect the binding
affinity or because there are a limited number of sites where
the label can react. Typically, only 8−10 fluorophores can be
conjugated to an antibody.
In general, amplifying the signal leads to enhanced
sensitivity. Various approaches to signal amplification have
been implemented. One approach is by using an enzyme that
generates many detectable signal molecules. Enzymatic turn-
over amplifies the signal and increases the sensitivity because
the number of detectable molecules is substantially higher than
the number of protein molecules captured. Commonly used
enzymes include alkaline phosphatase (AP), horseradish
peroxidase (HRP), and β-galactosidase. These enzymes turn
over many substrate molecules to generate many fluorescent
molecules. A second approach is to use nanomaterials.70−72
Nanomaterials have increased surface area such that they can
be conjugated to both an affinity reagent and many labels, such
as fluorophores or enzymes. Nanomaterials that are inherently
brighter, such as carbon dots73−75 or polymer dots,76−78 can
also be used as optical labels. Fluorescent silica nanoparticles in
which standard organic dyes are incorporated can also be used
to enhance the signal.79−81
Fluorescent molecules are sensitive to changes in the
chemical environment. Buffer composition, pH, and solvent
polarity can affect the quantum yield. Overlabeling an affinity
reagent with a fluorescent molecule can also change the
quantum yield due to quenching resulting from interactions
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between the dyes. Additionally, nonspecific binding can arise
between organic dyes containing aromatic groups and other
components in the assay such as surfaces.82 Such interactions
can be minimized by adding negative charges to the
fluorophore, such as PEG or sulfonate modifications, to
increase the hydrophilicity.15 Naturally occurring fluorophores
in a biological sample, such as aromatic amino acid residues,
NADH, and flavins, can also cause interference and increase
the background.
Other commonly used methods for optical detection,
particularly as part of the enzyme-linked immunosorbent
assay (ELISA), include electrochemiluminescence (ECL),
chemiluminescence, and colorimetric-based assays. In ECL,
an electron transfer reaction at the surface of an electrode
produces excited states that can then emit light. The MSD
assay uses ECL for protein detection and is discussed in
section 6.1.1.
Chemiluminescence is a result of a chemical reaction that
produces light. In a chemiluminescent reaction, oxidation of an
organic dye, such as luminol, with a strong oxidant, such as
hydrogen peroxide, in the presence of a catalyst produces a
light-emitting molecule. The emission lifetime of the molecule
can last from several seconds to hours. Commonly used
chemiluminescent reagents for protein detection are the
enzyme horseradish peroxidase and luminol or its derivatives.
Chemiluminescent detection does not require an excitation
source, and therefore, there is no background signal due to
excitation of other components in the sample matrix. A
disadvantage is that the generated chemiluminescent signal can
be relatively weak; therefore, approaches to enhance
chemiluminescent intensity have been developed.83 These
approaches include modification of luminol or addition of
various organic compounds, such as phenols, that enhance
chemiluminescence. Quantum dots and metallic nanoparticles
have also been used to enhance chemiluminescence.84−86 For
example, it has been shown that gold nanoparticles can catalyze
chemiluminescence of luminol.87 Additionally, carbon dots
have been shown to have chemiluminescent properties.88
Colorimetric signal generation is based on formation of a
colored precipitate, typically by an enzyme.89 Nanoparticle-
based nonenzymatic colorimetric detection has also been
demonstrated.90,91 Nanoparticle-based colorimetric assays can
exhibit high sensitivities for protein markers,92−95 down to the
attomolar range.96 Advantages of colorimetric assays include
relative simplicity, low cost, and ability to detect markers with
the naked eye.97
In addition to an optical label, optical detection tools often
require components such as a light source, optical filters, and
detectors, such as photodiodes, phototransistors, or photo-
multiplier tubes, which convert the incident light into an
electrical signal. Additional information on instrumentation for
optical detection can be found in the literature.68 Electro-
chemical methods for protein detection are widely used in the
literature and have been recently reviewed.98−100
3.5. Multiplexing
Measuring multiple different proteins in a single sample
simultaneously is advantageous for high-throughput analysis as
well as reduced time and cost. There are two commonly used
multiplex assay formats (Figure 6). The first format spatially
separates the capture reagents using a conventional microarray,
in which the identity of the capture reagent at each position in
the array is predetermined by its spatial location.101,102 For
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Figure 6. Approaches for multiplexing via (a) a conventional
microarray in which the identity of the target protein is known
based on the predetermined position of the affinity reagent and (b) a
suspension array in which the affinity reagents are attached to
encoded particles. Each optically distinct population of beads is
conjugated to capture antibodies to a specific protein. Printed with
permission from ref 108. Copyright 2006 John Wiley and Sons.
example, the SOMAscan assay, which is described in more
detail in section 6.2.1, can detect approximately 1300 different
proteins simultaneously using a nucleic acid microarray. The
second format employs encoded micro- or nanoparticles, in
which each uniquely encoded particle population is function-
alized with an affinity reagent specific to a given protein. There
are several ways to encode micro- and nanoparticles, including
optical encoding with fluorescent dyes or Raman tags.103−105
Luminex has developed a 100-plex assay by optically encoding
microspheres with combinations of fluorescent dyes.106,107
Other ways to encode are based on graphical, physical,
magnetic, and thermal properties. These methods have been
recently reviewed.108−110 Finally, encoding using nucleic acid
barcodes can provide high multiplexing capabilities.111
A major challenge with multiplexed protein detection assays
is nonspecific binding and cross-reactivity between affinity
reagents, secondary labels, and other components in the
biological sample.112 Despite many efforts, measuring multiple
protein analytes with high sensitivity, specificity, and accuracy
is still a major challenge.
4. CURRENT COMMONLY USED METHODS FOR
PROTEIN DETECTION
This section discusses the most widely used techniques for
protein measurements, particularly for life sciences research
and clinical diagnostics. Many of these techniques were
developed in the 1960s and 1970s and are still widely used
today. These techniques include direct detection of total
protein, the enzyme-linked immunosorbent assay (ELISA),
enzyme-linked immunospot (ELISPOT), Western blot,
protein microarrays, flow cytometry, proximity ligation assays
(PLAs) and other nucleic acid-based methods, mass
spectrometry, lateral flow assays, surface plasmon resonance
(SPR), and optical imaging. Improvements in protein
detection methods are commonly based on improving these
basic techniques. Other commonly used techniques, such as X-
ray crystallography, CD, NMR, and electron microscopy, will
not be covered. These techniques are used to analyze the
structure of a specific protein or interactions between two
proteins and require purified protein samples. This review
focuses on measuring unknown levels of proteins in a
biological sample. Several reviews have been recently written
on various aspects of protein detection techniques.113−120
4.1. Detection of Total Protein
Direct detection and quantification of total protein is
important for many applications. Methods using UV and
visible spectroscopy are commonly used for fast quantification
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of total protein concentration relative to a standard or based
on known protein extinction coefficients. The aromatic amino
acids, tryptophan and tyrosine and to a lesser extent
phenylalanine, absorb light at 280 nm, a property which
enables quantification based on absorbance. This technique is
easy to use, but disadvantages include interference from other
sample components, such as nucleic acids, and low sensitivity
due to low extinction coefficients of amino acid residues.
Additionally, different proteins have different absorbance
spectra depending on the structure and the number of
aromatic amino acid residues, which can complicate
quantification and reduce accuracy.
Another strategy for total protein quantification is based on
colorimetric protein assays. There are two general approaches.
The first approach is based on chelation of copper by proteins
and includes the biuret assay, the bicinchoninic acid assay
(BCA assay), and the Lowry protein assay. In the biuret assay,
peptides with three or more amino acid residues form a
colored chelated complex with Cu2+ under alkaline conditions.
The color intensity is proportional to the number of amino
acid residues. The biuret test is a simple and fast quantitative
test to measure total protein concentration, with low
sensitivities in the mg/mL range. The BCA assay, developed
in 1985, can be used to enhance sensitivity.121 It is based on
the biuret reaction, in which proteins first form a chelated
complex with Cu2+. Cu2+ is then reduced and chelated with
BCA to form a colored complex. Another widely used
technique is the Lowry test, which was developed in 1951.
The Lowry test is also based on the biuret reaction and
involves the reduction of copper (Cu2+ to Cu+) by proteins in
alkaline solutions and formation of a colored compound via
reduction of a color-enhancing Folin phenol reagent.122 The
colored compound is produced and can be measured at a long
wavelength (750 nm), which is advantageous since fewer
compounds absorb light at that wavelength and therefore the
background is lower.
The second approach is based on protein-binding dyes. The
most commonly used assay is the Bradford assay, developed in
1976, which utilizes the dye Coomassie G-250.123 The
Coomassie dye binds selectively to certain amino acids and
tertiary protein structures. Therefore, there can be variations
between dye-binding efficiency to different proteins, which can
reduce accuracy. For the dye to bind to proteins, the protein
mass must be high, and therefore, this method is generally used
for proteins with high molecular weights. The Bradford assay is
rapid, simple, and widely used, but its accuracy is limited.
Fluorescent dyes can also be used due to their favorable
properties including increased sensitivity, decreased back-
ground, and wider dynamic range. In this method, a
nonfluorescent dye interacts with proteins either covalently
or noncovalently. This interaction causes the dye to become
fluorescent. 1 2 4 For example, the CBQCA (3-(4-
carboxybenzoyl)quinoline-2-carboxaldehyde) assay is based
on binding of a nonfluorescent dye to primary amines of
proteins in the presence of cyanide or thiols. The binding
causes the dye to become fluorescent, and the fluorescence
intensity is proportional to the amount of total protein.
The methods described above vary in their ability to detect
and quantify different proteins, are subject to interference from
other molecules in the sample, and may require careful
calibration; therefore, the particular choice of protein assay is
highly dependent on the sample type.125 Nevertheless, these
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methods are advantageous for direct and simple detection and
quantification of total protein and are widely in use.
4.2. Enzyme-Linked Immunosorbent Assay (ELISA)
The most common tool for protein detection is the
immunoassay. The immunoassay was first introduced in
1960 to measure plasma insulin using radioactive labels.126
Due to safety concerns associated with the use of radioactive
labels, including the need for special facilities and disposal of
waste, alternative methods to replace radioactive labels were
necessary. In 1971, an enzyme-based immunoassay was
developed by two different groups independently in which
the radioactive label was replaced by an enzyme label.127
Engvall and Perlmann developed the enzyme-linked immuno-
sorbent assay (ELISA) for antibodies in rabbit serum using
alkaline phosphatase,128 and van Weemen and Schuurs
developed the enzyme immunoassay (EIA) for human
chorionic gonadotropin in urine using horseradish perox-
idase.129 In an ELISA, the enzyme label is used to amplify the
signal by catalyzing the formation of many thousands of
molecules of a detectable product. In this manner, each protein
binding event is amplified.
Currently, the enzyme-linked immunosorbent assay
(ELISA) is the gold standard tool for protein detection and
quantification. Commonly used enzymes include horseradish
peroxidase (HRP), alkaline phosphatase (AP), and β-
galactosidase. Many different substrates for these enzymes
have been developed, and the products can be detected using
various detection methods including colorimetric, fluorescent,
chemiluminescent, and electrochemical detection.
The main advantages of the ELISA include the ability to
measure proteins quantitatively with relatively high sensitivity,
in the pg/mL range, and with a wide dynamic range that spans
4 orders of magnitude. Although ELISAs are rather time
consuming to carry out because they involve relatively long
incubation times and extensive washing, they are easy to use.
ELISAs are widely used for both clinical diagnostics and basic
research. For example, ELISAs are commonly used in the clinic
to detect C-reactive protein (CRP), a protein that plays an
important role in activating the immune system.130 CRP levels
in the blood are often elevated in subjects with inflammation;
therefore, elevated CRP levels can be indicative of autoimmune
disease or bacterial infection. Another example in which
ELISAs are used in the clinic is for detecting antibodies against
HIV in blood. HIV antibodies are detectable in the blood 3
months following infection. However, it has been shown that
ultrasensitive immunoassays can detect HIV earlier than the 3-
month period. One example of such assay is the biobarcode
assay, which is discussed in more detail in section 4.7.131
Approaches to improve protein detection are primarily based
on improving the performance of ELISAs.
4.3. Enzyme-Linked Immunospot (ELISPOT)
The enzyme-linked immunospot (ELISPOT), which was
developed in 1983, is used to detect secreted proteins by live
cells in vitro.132 ELISPOT is conceptually similar to an ELISA.
Briefly, antibodies are coated onto the surface of a microtiter
plate. A sample containing cells that secrete proteins is added,
and the target protein then binds to the antibodies. A second
enzyme-labeled detection antibody is then added. The enzyme
produces a colored precipitate. The assay is easy to perform on
a large number of samples. This method is commonly used to
detect cytokine secreting cells. ELISPOT assays have been
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used to detect interferon gamma release for tuberculosis
diagnosis.133
4.4. Western Blot
The Western blot, which was developed by three different
groups independently between 1979 and 1981, is a widely used
technique in life science research to identify specific
proteins.134−136 In a Western blot, a sample is separated by
size using gel electrophoresis, transferred to a solid membrane,
blotted with a labeled antibody specific to the target protein,
and then visualized.137 Western blots can be used for
quantitative detection of proteins by using known standards.
A major advantage of Western blots is the added layer of
specificity, since proteins are initially separated by size. This
additional specificity contrasts with ELISA, which only detects
the label and does not provide any information on the
molecular size of the protein. Even though the Western blot is
rather time consuming and can only detect high protein
concentrations, it is widely used due to its high specificity and
simplicity, since it requires no complex instrumentation. A
variation of the Western blot is the dot blot, which is similar to
the Western blot but does not separate protein by electro-
phoresis. Recently, the single-cell Western blot was developed
for measuring proteins with high sensitivity on a single-cell
level. This method is described in section 6. The Western blot
is most widely used for basic research and is commonly used to
detect protein isoforms, truncated proteins, post-translational
modifications, and antibodies. Western blots are also used in
the clinic. For example, HIV diagnosis is typically a two-step
process in which the first step is detecting antibodies against
HIV in the blood using ELISA and the second step is
confirming the ELISA result using a Western blot.138,139 This
combined approach leads to 99% clinical sensitivity and
specificity.
4.5. Protein Microarrays
Protein microarrays are used for multiplexed detection of
proteins using reagents immobilized on a planar surface
typically made of silicon, glass, or nitrocellulose. Reagents can
be immobilized onto the surface of the microarray by various
approaches including contact printing or in situ synthesis of
reagents.140 There are two types of protein microarrays. The
first is based on a sandwich immunoassay (Figure 7A) in which
capture antibodies are immobilized onto the surface of the
microarray. The target protein first binds to the capture
antibodies, labeled with a detection antibody, and detected.
This type of microarray can be used for multiplexed detection
of proteins by immobilizing antibodies specific to different
proteins at known and predefined positions. In the second type
of microarray, also known as a reverse phase array (Figure 7B),
a sample is deposited onto the surface of the microarray and
the target protein is detected by probing with an antibody.
Reverse phase arrays are commonly used to detect protein
phosphorylation levels or antibodies. In protein microarrays,
the detected signal is usually fluorescent, chemiluminescent,
electrochemiluminescent, or colorimetric. Typically, the bind-
ing kinetics of protein microarrays are less favorable than the
binding kinetics of solution-based assays using microspheres
since the proteins must diffuse to the surface of the microarray
in order to bind to the capture reagent. This mass transport
limitation may result in reduced sensitives for protein
microarrays.
Protein microarrays are commonly used for protein
detection and are available commercially. One example is the
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Figure 7. Protein microarrays. (a) In a planar microarray, capture
antibodies specific to different proteins are immobilized at known and
predefined positions for multiplexed detection. Target protein binds
to the capture antibody and then to a labeled detection antibody.
Signal intensity is correlated with the presence and amount of the
target protein. (b) In a reverse phase protein microarray, the sample is
deposited onto the surface of the microarray and the target protein is
detected by probing with an antibody. Signal intensity is correlated
with the presence of the target protein. Modified and reprinted with
permission from ref 117. Copyright 2015 Nature Publishing Group.
ProtoArray from ThermoFisher. Protein microarrays can be
used to study protein−protein interactions and enzymatic
activity and for biomarker discovery.141 One of the earliest
implementations of protein microarrays was for analyzing the
biochemical activity of yeast proteins, including identifying
proteins that interact with calmodulin and phospholipids and
analyzing protein kinases.142,143 More recently, protein micro-
arrays were used for high-throughput detection of antibodies
against Plasmodium falciparum (Pf) in plasma before and after
malaria season.144 Additionally, protein microarrays have been
used for analyzing protein interactions with other molecules
including both nucleic acids145 and small molecules.146 Protein
microarrays are also used for clinical diagnostics.147 One
example is the Meso Scale Discovery (MSD) system. Another
example is the SOMAscan assay, which uses a nucleic acid
microarray for protein detection. These methods are further
described in section 6.
4.6. Flow Cytometry
Flow cytometry was first introduced in the 1960s and is used
to separate cells based on size and protein surface markers. In
flow cytometry, fluorophore-labeled antibodies bind to specific
cell-surface markers. The cells are then flowed through a flow
channel. Lasers are used to excite the fluorophores, and the
emission is measured at two different angles, providing the
ability to differentiate one cell at a time based on size and
surface markers. The main advantage of flow cytometers is
their high throughput, namely, their ability to analyze
thousands of cells per second. Another advantage is the ability
to sort using fluorescence-activated cell sorting (FACS) for
further downstream analysis of cells. Flow cytometry is
commonly used by immunologists, particularly to detect cell-
surface proteins known as clusters of differentiation (CDs).
For example, analysis of cell-surface markers using flow
cytometry was instrumental for characterizing B-cell lineage
development. Hardy et al. showed that progenitor and
precursor B cells in the bone marrow are a complex mixture
of cells at different stages of development.148 Flow cytometry
also has applications in clinical diagnostics of immunodefi-
ciency and hematopoietic disorders based on cell-surface
protein markers.149 In 2017, the FDA approved for the first
time a flow-cytometry-based assay for blood cancer diag-
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nostics.150 This assay detects several cell-surface markers in
peripheral blood, bone marrow, and lymph node samples for
diagnosis of leukemia, non-Hodgkin lymphoma, multiple
myeloma, myelodysplastic syndrome (MDS), and myelopro-
liferative neoplasms (MPN).
Other than cells, flow cytometers can also be used to detect
specific proteins using microspheres in a sandwich assay
format. Microspheres are coated with specific antibodies,
incubated with the target protein, and then labeled with a
second fluorescently labeled detection antibody. Flow
cytometry is becoming increasingly popular for protein
quantification due to the high multiplexing capabilities.
Fluorescent labels are now being replaced with metal ions in
a new technique known as mass cytometry. These new
techniques are discussed in more detail in section 6. The main
limitation of flow cytometry is high cost, the need for a skilled
operator, and relatively complex instrumentation.
4.7. Proximity Ligation Assay (PLA) and other Nucleic
Acid-Based Detection Methods
An approach for signal generation and amplification is to
convert protein binding into a nucleic acid signal.151,152 The
main advantage is that highly sensitive detection of nucleic
acids is possible because DNA can be amplified. By combining
the benefits provided by antibodies and the amplification
advantage provided by PCR, immuno-PCR was introduced
and enabled protein detection with greater sensitivity than the
conventional immunoassay. In this approach, the target protein
binds to capture antibodies immobilized on a surface. A
DNA−antibody conjugate then binds to the target protein.
The DNA is amplified by PCR and then detected by gel
electrophoresis.153 Further improvements to immuno-PCR
have been realized by using a sandwich assay consisting of a
capture antibody and a detection antibody that is conjugated
to an oligonucleotide label, amplifying the oligonucleotide
label and quantifying the label using qPCR.154 Another
method called the proximity ligation assay (PLA) is used for
protein detection (Figure 8).155,156 In PLA, two biotinylated
Figure 8. Proximity ligation assay (PLA). In PLA, the sample is first
incubated with antibodies conjugated to oligonucleotide probes (1).
Components for ligation and detection are added to the sample (2),
and quantitative PCR is used for detection (3). Modified and
reprinted with permission from ref 156. Copyright 2004 National
Academy of Science.
antibodies bind to different epitopes of the target protein
analyte. Two different streptavidin-labeled oligonucleotides are
bound to each of the two biotinylated antibodies. PLA is
designed to give a signal only when a protein is present due to
the proximity of the epitopes within the target protein. When
both oligo-labeled antibodies bind to the protein, a connector
oligonucleotide can hybridize to both of the oligonucleotides, a
ligation step is performed, resulting in a DNA template that
can be amplified and quantified using qPCR.156 A variation of
PLA utilizes rolling circle amplification (RCA) for single-
molecule detection using digital PCR that enhances the
sensitivity of the method.157 More specifically, two oligonu-
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cleotides complementary to the oligonucleotides connecting
the two antibodies are added and two restriction sites are
generated for digestion. The resulting DNA strands are
circularized by a second ligation and then amplified by RCA.
The amplified products are then detected by hybridization with
fluorescently labeled probes and counted using a microfluidic
device. An alternative to PLA is the proximity extension assay
(PEA), in which the DNA ligase is replaced by a DNA
polymerase. PEA aims to reduce the interfering effects of DNA
ligase in biological samples.158 Multiplexed PLAs have been
developed by using probes with different sequences and
lengths, allowing for development of a 24 plex assay.159 Both
PLA and PEA provide sensitive methods for protein detection
with subfemtomolar sensitivity using a low sample volume.
These assays can be used in solution-based assays to measure
protein levels and detect protein−protein interactions. They
can also be used for protein analysis in tissue samples. Nucleic
acid assays have been used for highly sensitive protein assays
with detection limits in the attomolar range.160,161 Recently,
new methods have been developed for DNA-based protein
detection, including use of DNA nanoswitches,162 digital
droplet PCR,163,164 and incorporation of nanoparticles.165
Another method for ultrasensitive protein detection is the
biobarcode assay developed in the Mirkin laboratory (Figure
9).160,161,166,167 First, antibody-functionalized magnetic par-
ticles bind to the target protein. Then gold nanoparticles,
which are functionalized with both detection antibodies and
oligonucleotide barcodes, bind to the target protein to form a
sandwich complex. The oligonucleotide barcodes are then
released. Prior to detection, the biobarcodes can also be
amplified by PCR. The biobarcodes hybridize to probes on a
microarray that are complementary to one-half of the
biobarcode sequence. Following hybridization to complemen-
tary probes on the microarray, nanoparticles functionalized
with oligonucleotides complementary to the other half of the
biobarcode sequence hybridize. Silver amplification is used to
enhance the signal, and gray spots can be detected using a
Verigene instrument. The assay results in high sensitivity in the
attomolar range. The biobarcode assay has been used for the
sensitive detection of prostate specific antigen (PSA) in the
blood of patients who have undergone radical prostatec-
tomy.168 Following radical prostatectomy, PSA is usually not
detectable using a conventional ELISA. Since increasing PSA
levels can be indicative of recurrence, high-sensitivity assays
such as the biobarcode assay can be used to detect recurrence
earlier.
Multiplexed detection can be achieved by using gold
nanoparticles each functionalized with a unique biobarcode
sequence (Figure 9).111 These unique biobarcodes hybridize to
complementary probes at predetermined positions on a
microarray. Another method that uses a DNA microarray for
multiplexed protein detection is the SOMAscan assay, which is
further discussed in section 6.2.1.
4.8. Mass Spectrometry
Mass spectrometry is a common tool for proteomics analysis. It
is probably the most comprehensive method for analyzing
complex mixtures because it provides a full profile of the
protein composition. Conventional tandem mass spectrometry
digests proteins into peptides, and these peptides can then be
used to derive the sequence of the original protein molecule.
This strategy, known as “bottom-up” proteomics, provides
invaluable information about proteins present in complex
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Figure 9. Biobarcode assay. Target protein binds to magnetic microparticles (MMPs) functionalized with capture antibodies. Each population of
nanoparticles (NP) is functionalized with unique biobarcodes and detection antibodies specific to a target protein. Nanoparticles bind to the target
protein. Biobarcodes are then released and hybridize to probes complementary to one-half of the sequence. Gold nanoparticles then hybridized to
the other half of the sequence. Silver is used to enhance the signal, and the signal intensity is measured. Modified and reprinted with permission
from ref 166. Copyright 2006 Nature Publishing Group.

samples.169 However, since the same peptide sequence may be

present in many different proteins, it is often challenging to
identify different proteins, such as those that arise from
different gene product and splice variants. Another strategy
that may overcome this challenge is “top-down” proteomics, in
which whole, intact proteins are identified directly using
tandem mass spectrometry. This approach enables analysis of
different types of proteins; however, it is still challenging to
analyze low-abundance proteins, and quantification is also
difficult. Additional information on mass spectrometry
proteomics techniques and advances is provided in other
reviews.12,13,170−173
4.9. Lateral Flow Assay (LFA)
Lateral flow assays (LFAs) are commonly used for clinical
diagnostics and point of care detection of proteins in various
sample matrices including blood and urine.174,175 The most
commonly used assay format is the sandwich assay. An LFA
assay consists of several zones, and a typical configuration is
shown in Figure 10. First, the sample is added to the sample
application pad. The sample then moves toward the conjugate Figure 10. Sandwich lateral flow assay (LFA). (Top) In a sandwich
pad, which houses an antibody conjugated to a label. LFA, the sample is first added to the sample application pad and then
Commonly used labels for LFAs are latex particles or gold migrates toward the conjugate pad, which houses antibodies
conjugated to a label. Protein binds to the labeled antibodies, and
nanoparticles, which can form colored precipitates. Next, the sample then moves to a band that contains immobilized capture
sample, which contains the protein bound to a labeled antibodies. Sandwich complex is then formed. Control band is
antibody, moves to a band that contains immobilized capture immobilized with antibodies that capture the labeled antibody to
antibodies. A sandwich complex is then formed. Often there is ensure the test is performing correctly. Excess reagents then migrate
a second control band, which is immobilized with antibodies past the bands and are trapped in the absorbent pad. (Bottom) Signal
that capture the labeled antibody, to ensure the test is intensity of the band is proportional to the concentration of the target
performing correctly. Excess reagents then migrate past these protein. Reprinted with permission from refs 175 and 177. Copyright
2016 Elsevier and 2009 MDPI.
bands and are trapped in the wick or absorbent pad. The signal
intensity of the band is proportional to the concentration of
the target protein. The results can be visualized with the naked which measures the presence of the protein human chorionic
eye or with a reader. Usually LFAs provide qualitative or gonadotropin (hCG) in urine.176
semiquantitative protein measurements. One limitation is that 4.10. Surface Plasmon Resonance (SPR)
when very high levels of protein target are present in the Surface plasmon resonance (SPR) is a label-free method that
sample, the antibody binding sites become saturated. This can be used for protein detection. SPR occurs when
results in a high-dose hook effect, which produces a lower conduction electrons undergo oscillations at metallic surfaces
signal intensity than expected. upon exposure to light.178,179 SPR detection of proteins is
LFAs are important for clinical diagnostics. A major based on immobilization of an affinity reagent onto a metal
advantage of LFAs is that they are cheap and rapid, with surface. The affinity reagent then specifically binds to the
results provided in minutes, do not require complex protein of interest in the sample. The binding event causes a
instrumentation, and are compatible with point of care measurable change in the refractive index. SPR intensity
applications. The most common use is the pregnancy test, depends on the dielectric properties of the medium as well as
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size, composition, and shape of the metallic structure.180
Additionally, SPR is highly dependent on the angle of
incidence of light, the wavelength of light, and the refractive
index of the sample. SPR is commonly used to study the
binding kinetics of protein−protein interactions, such as
binding of proteins to antibodies.181 Additionally, SPR has
been used to detect live malignant cells.182 Gold nanoparticles
conjugated to antiepidermal growth factor receptor (anti-
EGFR) antibodies were added to cultures of nonmalignant and
malignant cells. The anti-EGFR antibody-conjugated nano-
particles bound to the surface of the malignant cells with
substantially higher affinity than to the nonmalignant cells and
were detected by SPR. More recently, SPR has been used to
detect exosomes, nanometer-sized extracellular vesicles, based
on surface protein markers.183
SPR is advantageous due to its ability to generate
quantitative measurements with a lack of labeling variations
associated with optical labels such as fluorescent dyes.
Localized surface plasmon resonance (LSPR) of metal
nanostructures can be used to enhance the signal.184,185
Additionally, nanoparticle labels on detection antibodies in a
sandwich assay format can also be used to enhance the
signal.186,187
4.11. Optical Imaging
The subcellular localization of proteins is important for
function due to the unique chemical environment and
proximity to other interacting molecules. Delocalization of
proteins may result in a lack of function and disease. Therefore,
the spatial distribution of proteins in their native cellular
context is critical for understanding their function. Optical
imaging-based techniques are instrumental for studying
proteins in their native cellular environment.
Immunohistochemistry (IHC), first developed in 1941,188 is
used to detect proteins and their cellular localizations in tissues
and cells. Traditionally, a sample is first fixed or frozen; then
the antibody binds to the target protein and is then labeled to
produce a colored product that can be optically detected.
Antibody binding can be observed either by using a
fluorophore or an enzymatic reaction that produces a colored
product. IHC is a widely used technique for protein detection,
particularly in clinical diagnostics, where it is used for disease
diagnosis, prognosis, and treatment.189 Another similar
technique, immunocytochemistry (ICC), is used to analyze
proteins in cultured cells or cells in which the extracellular
matrix has been removed. Since sample preparation is different
for IHC and ICC, a given antibody may not be compatible
with both methods since the protein epitope may not be fully
exposed and cannot bind to the antibody. For IHC, a process
known as antigen retrieval is commonly used to unmask the
epitope by heating or digesting the sample with proteases. In
many cases a combination of both heating and digestion is
used. This process unmasks the protein epitope and allows the
antibody to bind to the protein.190
The main advantage of these techniques over other protein
detection methods is the ability to correlate the presence of a
protein with its location in a tissue or cell. This feature is very
important for studying cell function in normal and pathological
tissues and for differentiating between healthy and diseased
tissue. Recently, Thul et al. used immunofluorescence
microscopy to map the subcellular location of approximately
12 000 human proteins.191 Tissue microarrays, in which
hundreds of different tissue samples are assembled on a single
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slide, enable high-throughput analysis of many samples. Tissue
microarrays have been used for the Human Protein Atlas for
analysis of protein expression in normal and cancer human
tissue.1,192,193
Live cell imaging is becoming more popular to study protein
localization and dynamics in live cells. Since antibodies cannot
penetrate the cell membrane, proteins are genetically tagged
with fluorescent molecules, such as green fluorescent protein
(GFP). Other systems that are based on SNAP, CLIP, and
Halo tags have also been used.194
The study and detection of proteins in the context of their
cellular environment can be enhanced by improvements in
fluorescence microscopy. Super-resolution microscopy can
resolve features less than the diffraction limit of light, which
is about 200 nm in the lateral direction and 500 nm in the axial
direction. These techniques include stimulated emission
depletion (STED), photoactivation localization microscopy
(PALM), stochastic optical reconstruction microscopy
(STORM), structured illumination microscopy (SIM), satu-
rated structured illumination microscopy (SSIM), ground state
depletion (GSD), and reversible saturated optical (fluores-
cence) transitions (RESOLFT) and have been thoroughly
reviewed.195−198
5. CHALLENGES WITH PROTEIN DETECTION
The performance of a protein detection technique is assessed
by various parameters including sensitivity, specificity, dynamic
range, multiplexing capabilities, reproducibility, ease of use,
and cost. Currently, a major analytical challenge with protein
detection techniques is measuring multiple proteins, which are
present in complex biological samples, with high sensitivity and
a wide dynamic range. In this section, we will discuss some of
the current challenges, including sensitivity, specificity, and
multiplexing capabilities.
High-sensitivity measurements of proteins are particularly
important for many applications. One example is the study of
single cells. Single-cell transcriptomics has been widely used to
study heterogeneity between different cells and for under-
standing important biological processes such as stem cell
differentiation and cancer. However, our understanding of
protein levels and dynamics in single cells is limited.
Additionally, the relationship between mRNA levels and
protein levels on a single-cell level has been studied164,199−201
but is still not fully understood. Another example is
measurement of blood protein biomarkers for clinical
diagnostics, including disease diagnosis, monitoring, and
treatment. It is particularly challenging to detect low-
abundance proteins in the presence of high-abundance
proteins in the blood. Less than 10% of plasma proteins
have been quantitatively measured due to limitations in
analytical sensitivity.202,203 Therefore, high-sensitivity measure-
ments of proteins are critical.
The sensitivity, or detection limit, is typically calculated
based on the background signal of the assay.204,205 The
background signal is due to nonspecific binding of the affinity
reagents, labeling reagents, and components in the biological
sample with each other or with the surface. The detection limit
is usually calculated based on measuring a pure protein in a
buffer solution. However, many biological samples contain
interfering substances that may increase the background signal,
and therefore, it is important to assess the detection limit in the
biological sample matrix. To reduce nonspecific binding arising
from sample components, sample separation techniques can be
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employed including desalting, filtration, and affinity depletion measuring multiple proteins simultaneously with high
of other interfering molecules. As a result, the particular type of sensitivity. In this section, we will review techniques that are
sample is also important since different samples may contain currently being implemented to overcome limitations in both
different compositions of interfering substances. Another way sensitivity and multiplexing.
to overcome nonspecific binding is by optimizing buffer 6.1. Ultrasensitive Methods for Protein Detection
components, such as adding blockers. An important parameter
in protein detection is conversion of the target protein binding 6.1.1. Meso Scale Discovery (MSD). The Meso Scale
with the affinity reagent into a measurable signal. Discovery System (MSD) is based on an electrochemilumi-
Detection of a specific protein in a complex biological matrix nescent (ECL) sandwich immunoassay that can measure up to
is often challenging. Biological samples contain many protein 10 different proteins simultaneously (Figure 11).207
and nonprotein analytes that may interfere with detection of
the target protein. Some nonprotein target analytes may be
present at considerably higher levels than the target protein,
further complicating detection. Additionally, nontarget pro-
teins with similar structures may bind to the affinity reagents
and interfere with accurate quantification. High-affinity
reagents that are specific to the target protein and do not
bind to other nonanalyte molecules are necessary to enhance
specificity. Another challenge arises for some types of assays in
which an affinity reagent may specifically bind to a target
protein in a certain method but not in another. For example,
proteins are detected in their native form for sandwich
immunoassays and flow cytometry, but they are denatured in
Western blots and immunohistochemistry. Due to differences
in protein conformation and epitope structure, affinity reagents
that target the same protein may not be usable across different
methods. Therefore, detecting a specific protein target in a
biological sample is often quite challenging.
Another challenge with protein detection methods is
multiplexing. It is advantageous to simultaneously detect
multiple proteins in a single sample for increased throughput,
lower cost, reduced sample volume, and faster assay time. A
major challenge with multiplexed assays is cross-reactivity
between components in the assay, including affinity reagents
and other proteins, which often complicates detection and
Figure 11. Meso scale discovery (MSD) assay. (a) Generation of
accurate quantification. In many samples, the concentrations of electrochemiluminescence (ECL) based on oxidation of ruthenium at
different proteins in a biological sample can span several orders an electrode in the presence of tripropylamine (TPA). (b) ECL
of magnitude. Therefore, it is advantageous to have a wide ruthenium label used to modify antibodies. (c) MSD assay plate
dynamic range to measure both high- and low-abundance consisting of an injection-molded plate top and a bottom plate
proteins simultaneously in a single sample. A wide dynamic consisting of screen-printed carbon ink electrodes. (d) Well
range is also useful for measuring a single protein in different containing electrodes and antibodies. Each well contains 10 spatially
biological samples since some samples may have substantially defined positions that house capture antibodies specific to different
different levels of a given protein. Multiplexed assays also often proteins for multiplexed detection. (e) Sandwich immunoassay with
reduce the sample volume requirements. This feature is ECL detection. (f) Sector PR 400 plate reader. Reprinted with
permission from ref 207. Copyright 2007 Elsevier.
particularly important when sample volume is limited, for
example, when measuring proteins in cerebrospinal fluid,
tumor tissue samples, or blood obtained from infants. Electrochemiluminescence (ECL) involves a chemical
Therefore, a major analytical challenge is measuring multiple reaction that generates intermediates, which undergo an
proteins in a complex biological sample with both high electron transfer reaction at the electrode surface to produce
sensitivity and a wide dynamic range. excited states that emit light.208−211 Inorganic molecules, such
Finally, other analytical parameters of the assay, including as tris(2,2′-bipyridine)ruthenium(II) (Ru(bpy)32+), are widely
precision, accuracy, and reproducibility, are important. A final used to generate ECL. Due to its low molecular mass, many
consideration is whether a simple binary result, such as the Ru(bpy)32+ molecules can be attached to an antibody without
presence or absence of the target protein, is sufficient or if hindering the antibody’s ability to bind to the target protein.
quantitation is required. For detailed discussion of analytical Additionally, when a photon is emitted, the ground state is
parameters of immunoassays, the reader is referred to a regenerated, and therefore, a single label can undergo many
literature review.206 reaction cycles to produce numerous photons. Overall, signal
transduction using ECL is advantageous for several reasons
6. EMERGING METHODS FOR ULTRASENSITIVE AND including high sensitivity, wide dynamic range, simplicity, and
HIGHLY MULTIPLEXED MEASUREMENTS stability. A biological sample may contain molecules that are
Emerging new techniques have been developed to overcome autofluorescent and increase the background signal; therefore,
the challenges discussed in the previous section. A major a major advantage of ECL over fluorescence detection is the
analytical challenge with protein detection techniques is lack of a need for an excitation source. This results in lower
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Figure 12. Single-molecule array (Simoa) immunoassay. (a) Single protein molecule is first captured on beads, which are functionalized with
capture antibodies. Protein is then labeled with a biotinylated detection antibody and streptavidin-ß-galactosidase. (b) Excess beads are added to a
sample containing a low concentration of protein so that only one or zero protein molecules bind to each bead. In the presence of enzyme
substrate, beads are then loaded onto an array of femtoliter-sized wells such that only one bead can fit per well. Array is sealed with oil, and
fluorescence images of the array are then obtained. (c) Scanning electron micrograph image of a microwell array containing beads inside wells of
femtoliter volume. (d) Fluorescence images of a microwell array following generation of signal from a single enzyme molecule. Reprinted with
permission from ref 219. Copyright 2010 Nature Publishing Group.

background signals by eliminating detection of fluorescence
that is due to other components in the sample. Enhanced ECL
signals can also be generated, and several reviews have been
written on the topic.212−216
In the MSD assay, which is based on ECL detection, capture
antibodies are first screen-printed onto the bottom of a 96- or
384-well plate at predetermined positions. After binding of the
target protein to the capture antibody, a second detection
antibody that is labeled with ruthenium binds to the target
protein. When the ruthenium complex is placed at an oxidizing
electrode in the presence of tripropylamine (TPA), an ECL
signal is generated. The MSD assays have sensitivities in the
low pg/mL range and a wide dynamic range of 4 orders of
magnitude.217 MSD assays are commonly used to measure
protein biomarkers in blood for clinical applications.
6.1.2. Single-Molecule Arrays (Simoa). Digital ELISA
using single-molecule arrays (Simoa), which is being
commercialized by Quanterix Corp., is an ultrasensitive
method for protein detection using a bead-based sandwich
ELISA.218 In a conventional sandwich ELISA, the fluorescent
product of the enzyme−substrate reaction diffuses into a large
volume of approximately 50−100 μL. Therefore, millions of
enzyme-labeled immunocomplexes are required to generate a
measurable fluorescent signal above the background. In digital
ELISA, the fluorescent product of the enzyme−substrate
reaction is confined into femtoliter-sized wells, which are
termed single-molecule arrays (Simoa).219 Using this ap-
proach, the presence of a single-enzyme molecule can be
detected.220
In a Simoa immunoassay (Figure 12), antibody-coated
capture beads are added in excess to a sample containing low
concentrations of target protein molecules. On the basis of
Poisson statistics, either one or zero target protein molecules
will bind to each bead. To illustrate this concept, a 100 μL
amount of blood sample with 1 fM of the target protein
contains approximately 60 000 protein molecules. If 500 000
306
antibody-coated beads are incubated with the blood sample,
most of the beads will bind zero protein molecules while a
small number of beads will bind one protein molecule. A
negligible number of beads will bind more than one protein
molecule based on the Poisson distribution. The beads are
then incubated with a biotinylated detection antibody and
streptavidin-ß-galactosidase, forming an enzyme-labeled im-
munocomplex. The beads are then loaded onto an array of
femtoliter-sized wells, in which each well is physically able to
hold exactly one bead. The wells are filled with a fluorogenic
substrate and sealed with oil. Beads containing a single
immunocomplex will result in enzymatic conversion of many
substrate molecules into product. Because the fluorescent
product is confined to an extremely small volume of
approximately 50 fL, a high local concentration of fluorescent
product results that is easily detected with an imaging detector.
Hundreds of thousands of wells are interrogated simulta-
neously, and the ratio of beads containing an enzyme label to
the number of total beads in wells corresponds to the target
protein concentration in the original sample. In this manner,
single-protein molecules can be detected using a digital
readout. A wider dynamic range beyond the digital range can
be obtained by using the average fluorescence intensity of the
active beads. This results in a dynamic range that spans over 4
orders of magnitude.221 Simoa immunoassays have been
developed for multiplexed detection of six different proteins
using dye-encoded beads.222 Simoa immunoassays have been
used for ultrasensitive detection of biomarkers with applica-
tions in neurology,223 cardiology,224 oncology,225,226 and
immunology.227
6.1.3. Single-Molecule Counting (SMC). The single-
molecule counting (SMC) platform, also known as the Erenna
immunoassay system from Singulex Inc., is capable of high-
sensitivity protein measurements with detection limits in the
sub pg/mL range.228,229 SMC is based on a traditional
sandwich immunoassay, in which a capture antibody
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immobilized on the surface of either a microtiter plate or
magnetic beads binds to the target protein. A second detection
antibody that is conjugated to a fluorophore then binds to a
different epitope on the target protein, forming a sandwich
complex. Following several washing steps, the fluorescently
labeled detection antibody is eluted from the immunocomplex
into a small volume. The eluate is detected using a laser in a
small interrogation area. As the fluorescently labeled detection
antibodies flow through the interrogation area, they are
detected as positive or negative events using a confocal
imaging system. The emitted photons are counted over defined
time intervals, and if the photon count is sufficiently higher
than the background, the signal is considered positive. By
counting the number of positive events, a digital readout is
obtained. At higher protein concentrations, an analog mode is
used in which the total number of photons is used. This
provides a wide dynamic range of approximately 4 orders of
magnitude. The original system was designed for single-plex
assays; however, recently, a three-plex assay was developed
using three labels and three different lasers.230
6.1.4. Single-Cell Western Blot. A sensitive protein
detection method for one specific application is the single-cell
Western blot (Figure 13).231,232 Many protein detection
Figure 13. Single-cell Western blot. First, single cells settle into wells
in a microwell array made of a photoactive gel. Dimensions of the
wells are such that only one cell can fit inside each well. Cells are then
lysed with a denaturing RIPA buffer. After polyacrylamide gel
electrophoresis (PAGE), the proteins are immobilized onto the gel
with UV light followed by probing with a primary antibody (1° Ab)
and then secondary labeled antibody (2° Ab*). Fluorescent images of
the array are obtained to detect the target protein. Chemical stripping
and reprobing enables detection of multiple proteins. Modified and
reprinted with permission from ref 233. Copyright 2014 Nature
Publishing Group.
methods use antibodies to capture a specific protein. While
antibodies are enabling for protein detection, limitations exist
due to antibody specificity and cross-reactivity with other
similar proteins. The Western blot, one of the most commonly
used techniques for protein detection, first separates proteins
based on molecular mass using gel electrophoresis prior to
antibody probing and is thus more specific for the target
protein. In the traditional Western blot, thousands of cells are
needed to overcome the background signal. There is increasing
interest in analyzing proteins on the single-cell level, for
example, for identification of rare circulating tumor cells
(CTCs) in the blood. Microfluidic devices offer an advantage
by reducing the amount of required sample, facilitating
separation of rare CTCs from the more abundant red and
white blood cells. However, a major challenge is downstream
analysis of the isolated CTCs on a single-cell level.
The single-cell Western blot addresses some of these
limitations. The device is comprised of a microscope slide
and a layer of polyacrylamide gel that is patterned with an array
of microwells, in which each well can physically hold only a
single cell. A single cell is loaded onto the microwell array and
307
Review
allowed to settle into a well. The cells are then lysed. The
proteins are loaded electrophoretically into the surrounding
gel, and all of the Western blot steps including electrophoresis,
blotting, and probing are performed. This technique has
several advantages including the ability to assay low volumes
and low concentrations of proteins. An additional advantage is
the ability to integrate antibody probing with gel electro-
phoresis. This method allows detection of multiple protein
isoforms as well as truncated proteins, which may be of
importance for disease detection. Using this approach,
multiplexed detection of 12 different proteins has been
accomplished.232
6.2. Highly Multiplexed Methods for Protein Detection
6.2.1. SOMAscan Assay. Oligonucleotides containing
modified nucleic acid side chains can bind to proteins with
high affinity. Various side-chain modifications for deoxyuridine
triphosphate (dUTP) have been developed to enhance binding
affinity. These modifications mimic the hydrophobic amino
acid residues tryptophan and phenylalanine, which are found in
many protein−protein binding regions. The modified aptamers
can achieve high affinities, with dissociation constants of less
than 1 nM. These high-affinity reagents are termed slow off-
rate-modified aptamers, abbreviated as SOMAmers.234,235 On
the basis of these SOMAmers, SomaLogic developed the
SOMAscan assay for highly multiplexed protein detection. The
principles of the assay are shown in Figure 14. For protein
detection, SOMAmers are designed with three tags: a
fluorophore, a photocleavable linker, and biotin. The
SOMAmer reagents first bind to beads functionalized with
streptavidin via biotin−streptavidin interaction. Then the
beads are incubated with the sample that contains the target
protein, and the target protein binds to the SOMAmer reagent
attached to the beads. The unbound proteins are washed away,
and then the proteins bound to the SOMAmer are tagged with
biotin. Ultraviolet light is then used to break the photo-
cleavable linker and release the SOMAmer reagent. Some of
the released SOMAmer reagents are associated with a
biotinylated target protein. A polyanionic competitor is then
added to further reduce nonspecific binding. Streptavidin-
functionalized beads are then used to recapture the
SOMAmer-biotinylated target protein complex. Additional
washing steps remove nonspecifically bound SOMAmers. The
SOMAmer reagent is then released in a denaturing buffer and
hybridized to an array of single-stranded complementary DNA
probes. Fluorescence intensity is then related to the protein
levels in the sample. The SOMAscan assay has been used for
biomarker discovery, primarily in biological samples such as
blood.236
The main advantage of the SOMAscan assay is its high
multiplexing capabilities. SomaLogic has generated SO-
MAmers specific to approximately 1300 human proteins with
sensitivities in the low-picomolar to high-femtomolar
range.237,238 A major goal of the company is to develop
additional SOMAmers that recognize many different proteins
to enable proteomics-level measurements. However, since the
technology is still in its early stages, additional analytical
variables must be determined, such as cross-reactivity and assay
variability.239 Nevertheless, this platform enables highly
multiplexed protein measurements with sufficient sensitivity
for unbiased protein measurements.
6.2.2. Luminex. The Luminex xMAP system is based on a
sandwich immunoassay format that incorporates flow
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Figure 14. SOMAscan assay. (a) SOMAmers, which are functionalized with a fluorophore, photocleavable linker, and biotin, are immobilized onto
streptavidin (SA)-coated microspheres and incubated with a sample. (b) Target protein binds to the SOMAmer reagents. (c) Target protein is
tagged with biotin. (d) UV light is used to cleave the photocleavable linker on the SOMAmer and release the SOMAmer−protein complex from the
microspheres. (e) Addition of a buffer containing a polyanionic competitor disrupts nonspecific binding. (f) SOMAmer−protein complexes are
captured on a new set of streptavidin-coated microspheres via the biotin on the target protein. (g) Denaturing buffer is used to release the
SOMAmers from the microspheres. (h) SOMAmers hybridize to complementary probes on a microarray and are detected by fluorescence.
Fluorescence intensity is correlated to the target protein concentration. Reprinted with permission from ref 236. Copyright 2014 Elsevier.

Figure 15. Mass cytometry (CyTOF). Antibodies labeled with heavy isotopes bind to the target proteins. Each cell is nebulized and introduced into
an inductively coupled plasma (ICP) and ionized. Abundant ions are removed, and remaining heavy elements are analyzed. Signal of each isotope
label is correlated to the presence of the target protein. Reprinted with permission from ref 244. Copyright 2012 Cell Press.

cytometry as the detection platform. Luminex assays can
achieve high multiplexing capabilities of 100 analytes by
entrapping different ratios of dyes into microspheres to obtain
optically distinct populations. Each optically distinct popula-
tion of microspheres is attached to capture antibodies specific
to a given protein. Following binding of the target protein, a
second antibody conjugated to phycoerythrin (PE) binds to
the protein. The microspheres are then analyzed using a flow
cytometer to detect the internal fluorescent labels to identify
the microsphere’s specificity and measure the PE emission
308
intensity corresponding to the amount of target protein bound
to the bead. A major advantage of the Luminex system is its
high-multiplexing capabilities. However, commercially avail-
able kits typically do not exceed simultaneous measurements of
more than 30 proteins due to cross-reactivity. The sensitivity of
Luminex assays is generally between 1 and 10 pg/mL range,
and the dynamic range spans approximately 3−4 orders of
magnitude. The Luminex system is widely used to measure
proteins for various research and approved in vitro diagnostics
(IVDs) applications including infectious diseases, human
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leukocyte antigen (HLA) detection, and more.240 Another 7.1.1. Upconverting Nanoparticles. Lanthanides exhibit
similar platform is the cytometric bead array (CBA), a bead- anti-Stokes luminescence or upconversion luminescence.250
based sandwich immunoassay based on flow cytometry, that is Upconversion is a nonlinear optical process in which
used for multiplexed detection of proteins.241,242 absorption of two or more photons leads to emission of a
6.2.3. Mass Cytometry (CyTOF). Mass cytometry was photon at a shorter wavelength. Upconverting nanoparticles
developed in 2009 to allow for protein detection on a single- can be used in a sandwich assay for protein detection, in which
cell level.243 Mass cytometry, also known as CyTOF, combines the detection antibody is labeled with an upconverting
the principles of flow cytometry and atomic mass spectrom- nanoparticle. The label is excited in the infrared region and
etry, enabling analysis of 40 parameters per cell with very high emits in the visible region for detection (Figure 16).251
throughput.244,245 The work flow of single-cell mass cytometry
(Figure 15) includes coupling antibodies to a stable non-
radioactive rare-earth metal isotope. The labeled antibodies
bind to the target protein of interest. The labeled cell is then
nebulized into a droplet and introduced into an inductively
coupled argon plasma (ICP). Once the cell enters the plasma,
the sample is vaporized and the cellular material in the droplet
is ionized, including the abundant isotopes in the cell such as
carbon, nitrogen, and oxygen. The ionized sample is passed
through a mass filter, which allows only the heavy reporter ions
through, and these ions are analyzed by the mass spectrometer.
The mass spectrometer then measures the composition of the
metal isotopes. This number is correlated to the abundance of
antibody bound targets in the original sample. Mass cytometry
has been used for various applications. Examples include Figure 16. Upconverting nanoparticles. Absorption of near IR light
single-cell analysis of immune and drug responses246 and (NIR) and energy transfer lead to upconverted blue, green, or red
analysis of T cells in healthy and HIV-infected subjects.247 emissions. Reprinted with permission from ref 251. Copyright 2009
Recently, metal isotopes have been used for multiplexed John Wiley and Sons.
detection of proteins in tissue samples.248,249
The main advantage of mass cytometry is the ability to Upconverting nanoparticles have several favorable optical
overcome the need for fluorophores by labeling antibodies properties including high stability and quantum yield. Addi-
with rare-earth heavy metals. The use of heavy metals provides tionally, background arising from absorbing molecules in a
an advantage for multiplexed detection since there are about biological sample is low since most biomolecules in a sample
100 isotopes that are potentially available for mass cytometry absorb in the UV region and not in the IR region.
and only 18 fluorophores for flow cytometry due to the Upconverting nanoparticles have been used as optical labels
spectral overlap of fluorophores. Additionally, the signal from for multiplexed detection and optical encoding.252−254 Several
the heavy rare-earth labels has low background because there reviews have been written on their properties and applica-
are no rare earths present in biological samples. Therefore, the tions.255−262
measured signal is highly specific to the rare-earth labels. This 7.1.2. Photoelectrochemical Detection (PEC). In
contrasts with optical detection of fluorophores, in which the photoelectrochemical (PEC) signal transduction, light is used
biological sample may contain molecules that are autofluor-
to excite photoactive species, which produce a measurable
escent and interfere with detection of the fluorescent label.
electrical signal (Figure 17). Since the excitation source
Another advantage of mass cytometry is its very high
(optical) is different than the detected signal (electrical), PEC-
throughput, with an ability to assay millions of single cells.
The main disadvantages of the method are that it can provide
only relative quantification and has low sensitivity. Several
other disadvantages are a lack of ability to sort the cells since
they are vaporized, challenges with quality control, statistical
analysis of complex data, and limited availability of commercial
rare-earth metal-labeled antibodies.

7. ADVANCES FROM THE LITERATURE

7.1. Advances in Labeling and Signal Detection
A major consideration for protein assays is converting the
binding of the target protein to an affinity reagent into a
measurable optical, electrochemical, or mechanical signal. In
general, enhancing the signal leads to higher sensitivities.
Various approaches to enhance the signal of labeling reagents
have been developed. In this section, we discuss several novel Figure 17. Schematic of photoelectrochemical detection of proteins.
and promising approaches for labeling and signal detection, Affinity reagent immobilized on the surface binds to the target
including upconverting nanoparticles, photoelectrochemical protein, resulting in steric hindrance that decreases the intensity of the
detection, optical ring resonators, and surface-enhanced photocurrent. Reprinted with permission from ref 265. Copyright
Raman scattering (SERS) tags. 2015 American Chemical Society.

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based signal detection could have potentially high sensitivity
due to reduced background.263−266 Therefore, even if bio-
logical molecules in the sample are excited, they will likely not
be detected. There are two essential components of PEC
assays for protein detection: an affinity reagent that is near the
signal transducer and a photoactive species. Typical molecules
used for PEC signal generation are organic or inorganic
semiconductors, quantum dots,267,268 or secondary enzyme
labels, such as glucose oxidase, horseradish peroxidase, alkaline
phosphatase, and acetylcholine esterase, that can produce
photocurrent due to catalysis.
Development of PEC-based assays for protein detection has
gained momentum in recent years. Two general approaches
have been used. The first is label-free detection in which
binding of the target protein to an affinity reagent results in
steric hindrance that decreases the intensity of the photo-
current.269,270 The second approach is based on a typical
sandwich-assay format in which an affinity reagent binds to the
target protein, and subsequent labeling with a PEC active
species results in measurable photocurrent. High-sensitivity
PEC protein assays with detection limits in the subpg/mL
range have been developed.271 Recently, multiplexed protein
detection methods based on an antibody array or two different
enzyme labels that produce distinguishable PEC signals were
developed.272−274 PEC assays are an emerging class of protein
assays due to their potential for high sensitivity and relative
simplicity.
7.1.3. Optical Ring Resonators. In an optical ring
resonator, light undergoes total internal reflection along a
curved boundary in a structure known as an optical
microcavity, resulting in light propagation in circulating
waveguide modes or whispering gallery modes (WGMs).
The sensing layer is modified with affinity reagents, and when
the target protein binds there is a shift in the resonance
wavelength. Figure 18 shows a schematic of WGM for protein
detection. WGM can have high sensitivity capable of detecting
Figure 18. Whispering gallery mode (WGM) sensor for protein
detection. (a) Resonance shift is associated with protein binding. (b)
WGM in a dielectric sphere. Light circumnavigates the glass sphere.
Antibodies are immobilized onto the surface, and binding of the
protein results in a shift of the resonance wavelength. (c) Single-
protein molecule binding events are theorized to appear as steps in
the wavelength shift. Reprinted with permission from ref 275.
Copyright 2008 Nature Publishing Group.
310
Review
single molecules.275−278 Additionally, multiplexed detection for
five protein analytes has been demonstrated using arrays of
microring resonators.279 Additional information on WGM is
provided in refs 280 and 281.
7.1.4. Surface-Enhanced Raman Scattering (SERS)
Tags. A molecule in a sample can be identified based on its
Raman spectra. Raman spectroscopy on its own is not
sufficient to detect protein molecules in most cases because
Raman signals are weak. Therefore, surface-enhanced Raman
scattering (SERS) is often used in which a metal surface,
generally composed of gold or silver, enhances the Raman
spectral lines. SERS tags, metal nanoparticles composed of a
Raman reporter that has strong Raman scattering, a protective
shell, and an affinity reagent, have been used to enhance the
sensitivity of Raman-based protein assays. SERS tags have
several advantages including high sensitivity,282 spectral lines
with narrow line widths, and multiplexing capabilities.
Furthermore, SERS tags have high stability and do not
photobleach. SERS tags can be detected with NIR lasers to
minimize background autofluorescence arising from compo-
nents in a biological sample.283 A multiplexed sandwich assay
using SERS tags as labels is shown in Figure 19.284 Different
approaches have been developed for protein detection using
SERS.285−287 Several excellent reviews have been written on
use of SERS tags and bioassays.288−291
7.2. Miniaturization
Miniaturized assays are advantageous due to size compatibility
between the target protein and the assay format. Major
advantages of these systems are low volume requirements and
enhanced sensitivity. In this section, we discuss miniaturized
approaches for protein detection including mechanical
detection using microcantilever arrays and ultrasmall contain-
ers.
7.2.1. Microcantilever-Based Assays. Advances in
micro- and nanofabrication technologies have enabled develop-
ment of miniaturized mechanical-based assays for protein
detection in which binding of a protein analyte to an affinity
reagent leads to a detectable change in mass.292,293 Mechanical
assays have several advantages for protein detection. They can
achieve yoctogram resolution,294 with single-protein molecule
detection,295 are amenable to multiplexing, are low cost, and
can be used for point of care applications.296 Additionally, they
are available in a label-free assay format but can also be
integrated with secondary labeled affinity reagents to enhance
the signal.
Recently, microcanteliver assays have been used for sensitive
and multiplexed protein detection based on mechanical signal
transduction.297−300 In such assays, an affinity reagent
immobilized on the surface of the microcantilever specifically
binds to the target protein, resulting in a shift of the resonance
frequency or lateral sheer stress that leads to bending of the
microcantilever.301 The resonance frequency and bending can
be measured using piezoresistivity or optical deflection of a
light beam at the surface of the microcantilever. A scheme is
shown in Figure 20.302
Microcantilevers have several advantages for protein
detection One advantage of microcantilever arrays is their
ability for multiplexed detection (Figure 20B).303−305 Addi-
tionally, microcantilever assays have a fast response time and
low cost. They are also amenable to microfabrication
technologies and integration into portable point of care
devices. Microcantilevers can be used for highly sensitive and
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Figure 19. Schematic of multiplex protein sandwich assay on a porous hydrogel bead using SERS nanotags. Porous hydrogel beads are modified
with capture antibodies, which bind to the target protein. Target protein is then labeled with SERS nanotags which are functionalized with a
detection antibody. Raman dyes are embedded in the Au core and the Ag shell. Two different SERS nanotags have been used for multiplexed
detection. Reprinted with permission from ref 284. Copyright 2018 American Chemical Society.

Figure 20. Microcantilever for protein detection. (a) Affinity reagents are immobilized onto the surface of the microcantilever. Antifouling
molecules are coated onto the bottom surface of the microcantilevers to reduce nonspecific binding. Target protein then binds to the affinity
reagent. Secondary gold−nanoparticle functionalized with antibodies binds to the protein. (b) SEM of the microcantilevers. (c) Optical beam
deflection for measuring microcantilever vibration. (d) Mass loading onto the microcantilever results in a shift in resonance frequency. Reprinted
with permission from ref 302. Copyright 2014 Nature Publishing Group.

quantitative detection of proteins.306−308 Improvements in
sensitivity using secondary antibodies tagged with nano-
particles, which increase the mass response of the microcanti-
lever assays, have resulted in sensitivity in the low fg/mL
range.302 An important consideration is nonspecific binding at
the bottom surface of the microcantilever. Surface passivation
can be used to overcome this limitation.309
7.2.2. Ultrasmall Containers. Miniaturized systems are
favorable for protein detection due to the compatibility
between the target protein molecule and the size of the
311
container. Protein molecules are a few nanometers in size and a
single cell about a few micrometers in size. Analysis performed
in a test tube, in which a single protein molecule or a single cell
is present in a large volume, will likely result in inability to
locate, capture, and detect the protein molecule or cell.
Confinement of molecules into ultrasmall containers enables
detection of proteins at the single-molecule and single-cell
level.
Several approaches using ultrasmall volume containers for
protein analysis have been developed. One example is the
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Chemical Reviews Review

Figure 21. Single-molecule enzyme analysis using a microwell array. (a) Enzyme F0F1 ATPase pumps protons from the outside to the inside of the
well by hydrolyzing ATP. Fluorescent pH indicator is present inside the well and monitors changes in pH. (b) Fluorescence images after addition of
ATP at 0 and 6000 s. Right panel (blue) shows the difference in intensity between the two time points. Reprinted with permission from ref 311.
Copyright 2014 Nature Publishing Group.

single-molecule array (Simoa) immunoassay, which was

discussed in section 6.2.2. In this technique, the fluorescent
product of the enzyme−substrate reaction is confined to a
small volume of approximately 50 fL. Another example is the
SlipChip immunoassay in which a bead-based immunoassay is
performed in nanoliter volumes.310 Finally, ultrasmall contain-
ers can also be used to measure the enzymatic activity of
proteins. For example, this approach was used to study the
transporter activity of F0F1 ATPase, which pumps protons
across a membrane by hydrolyzing ATP (Figure 21).311 In this
method, a fluorescent pH indicator is first added to the wells
on a microwell array. The microwell array is then coated with a
lipid bilayer, which mimics the lipid bilayer of a cell. Liposomes
embedded with zero, one, or two molecules of the enzyme
F0F1 are then introduced onto the array, and some fuse with
the lipid bilayer on the microwells. ATP is then added to
initiate the proton-pumping enzymatic activity of F0F1 ATPase
across the lipid bilayer. Fluorescent images are then obtained
to detect single-molecule activity. Another example is the
Nanopore technology, which can be used to detect single-
protein molecules.312 In this method, changes in ionic current
when the protein moves into the pore are monitored.
Nanopores have been used to distinguish between different Figure 22. Highly multiplexed detection of secreted proteins from
phosphorylation states. single cells. Single cells are first captured inside a microwell array.
Ultrasmall containers have also been used for single-cell Following incubation, secreted cytokines are captured onto capture
analysis. One application is detection of cytokine release from antibodies. Barcoded slide is then removed, and a sandwich assay is
single cells using an array of wells in which only one cell can fit performed. By using three optical encodings and 15 spatial encodings,
per well.313 Using this approach, highly multiplexed detection a 45-plex assay is possible. Modified and reprinted with permission
of secreted proteins from single cells was developed (Figure from ref 315. Copyright 2015 National Academy of Sciences.
22).314,315 In this method, single cells are first isolated inside
individual microchambers. Fifteen capture antibodies specific droplet digital PCR (ddPCR) for mRNA quantification
to different cytokines are coated onto the surface of the (Figure 23).164 Single cells are isolated and then lysed, and
chamber at known and predefined positions. Following a 12− the cell lysate is split into two separate samples. For digital
24 h incubation, the capture antibodies bind to the secreted PLA, the cell lysate sample is incubated with oligonucleotide-
cytokines. The barcoded chamber is then removed, and a bound antibodies. The antibodies bind to the target protein,
sandwich assay is performed by using three different optical bringing the oligonucleotides near each other. A connector
labels. Using a combination of spatial and spectral encoding, a oligonucleotide is added that hybridizes to the probes to form
42-plex assay was developed. A second approach based on a complex consisting of the target protein analyte, the probes,
isolating single cells inside microwells is the single-cell Western and connector oligonucleotide. Ligation is performed, forming
blot, which was previously described in section 6.1.4. double-stranded DNA (dsDNA) followed by proteolytic
Another method used to quantify proteins and mRNA levels digestion, resulting in a solution containing the dsDNA.
simultaneously in single mammalian cells is based on a digital Using a commercially available device for ddPCR (BioRad),
proximity ligation assay (PLA) for protein quantification and the solution containing the dsDNA is diluted and emulsified to
312 DOI: 10.1021/acs.chemrev.8b00257
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Chemical Reviews
Figure 23. Digital Droplet PCR for quantification of proteins and mRNA in single cells. Single cells are isolated and then lysed, and the cell lysate is
split into two separate samples. For protein detection, PLA is performed followed by proteolytic digestion, resulting in a solution containing
dsDNA. Solution containing the dsDNA is diluted and emulsified to form about 20 000 nL droplets, such that each droplet contains either 1 or 0
dsDNA molecules. Each dsDNA molecule is then amplified by PCR, and digital counting is performed by measuring the fluorescence using a
droplet reader. For mRNA detection, digital droplet PCR is performed. In this manner, both protein and mRNA from a single cell can be measured.
Reprinted with permission from ref 164. Copyright 2016 Cell Press.
form about 20 000 nanoliter droplets, such that each droplet
contains either one or zero dsDNA molecules. Each dsDNA
molecule is then amplified by PCR, and digital counting is
performed by measuring the fluorescence using a droplet
reader. Digital PLA using ddPCR is combined with RT-ddPCR
for simultaneous quantification of both protein and mRNA
from a single cell. For protein quantification, femtomolar
sensitivity corresponding to approximately 10 000 molecules
was achieved with a dynamic range of about 3 orders of
magnitude. For mRNA quantification, a limit of detection of
approximately 52 mRNAs per cell was achieved.
8. CONCLUSION AND FUTURE DIRECTIONS
In this review, we discussed the wide variety of approaches for
protein detection. Several parameters, including sensitivity,
specificity, dynamic range, multiplexing capabilities, reprodu-
cibility, ease of use, and cost, are important in protein
detection techniques. A major analytical challenge with current
protein detection techniques is measuring multiple proteins
that are present in complex biological samples with high
sensitivity and a wide dynamic range. Strategies to overcome
these challenges have been developed; however, there is still a
trade-off between high-sensitivity measurements and highly
multiplexed measurements of proteins. As a result, it is often
necessary to decide whether it is more advantageous to detect
several specific proteins using a candidate-based, hypothesis-
driven approach or to detect as many proteins as possible using
an unbiased, high-throughput approach that is less sensitive.
A major challenge in ultrasensitive and highly multiplexed
protein detection is nonspecific binding between biomolecules
in the sample, assay reagents, and surfaces. Nonspecific binding
leads to background signals that obscure target protein
binding. Surface passivation methods that reduce nonspecific
binding as well as development of high-affinity reagents that do
not cross-react will enhance the performance of protein
detection methods. In addition, low-cost protein detection
methods for applications in developing countries or point of
care clinical applications are still necessary.
Nevertheless, major advances in protein detection methods
have been developed in recent years. One example is the
SOMAscan assay, which is capable of highly multiplexed
313
Review
protein measurements. Ultrasensitive methods such as the
Meso Scale Discovery assay (MSD) and single-molecule arrays
(Simoa) are being used to detect previously unmeasurable
levels of proteins. Recent advances from the literature, such as
novel optical labels and assay miniaturization, enable protein
detection on the single-molecule and single-cell level. These
assays will undoubtedly lead to interesting new discoveries in
biology as well as new clinical diagnostics applications.
AUTHOR INFORMATION
Corresponding Author
*E-mail: dwalt@bwh.harvard.edu.
ORCID
Limor Cohen: 0000-0003-1448-0925
David R. Walt: 0000-0002-5524-7348
Notes
The authors declare the following competing financial
interest(s): David R. Walt is the scientific founder and a
board member of Quanterix Corporation. All other authors
declare no competing financial interest.
Biographies
David R. Walt is Professor of Pathology at Harvard Medical School
and Brigham and Women’s Hospital, a Core Faculty Member of the
Wyss Institute at Harvard University, and a Howard Hughes Medical
Institute. He is the Scientific Founder of multiple life sciences
companies. He has received numerous national and international
awards for his work in the field of optical microwell arrays and single
molecules. In addition to his lab’s fundamental work on single
molecules, his lab develops new diagnostics technologies and applies
them to pressing clinical problems. He is a member of the National
Academy of Engineering, the National Academy of Medicine, and a
Fellow of the American Academy of Arts and Sciences, the American
Institute for Medical and Biological Engineering, and the National
Academy of Inventors.
Limor Cohen is a Ph.D. candidate in the Department of Chemical
Biology at Harvard University. She received her B.A. degree in
Chemistry from New York University in 2012. Her research interests
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Chemical Reviews
lie in the development of analytical tools for ultrasensitive detection of
proteins and nucleic acids along with clinical applications.
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321 DOI: 10.1021/acs.chemrev.8b00257

Chem. Rev. 2019, 119, 293−321