Biosensors Electrochemical 2011

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Expert Opin Med Diagn. Author manuscript; available in PMC 2012 September 1.
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Expert Opin Med Diagn. 2011 September 1; 5(5): 381391. doi:10.1517/17530059.2011.607161.
Magnetic particles in ultrasensitive biomarker protein

measurements for cancer detection and monitoring
Vigneshwaran Mani, Bhaskara V. Chikkaveeraiah, and James F. Rusling,,*
Department of Chemistry, University of Connecticut, 55 North Eagleville Road, Storrs,
Connecticut 06269
Department
of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut
06032
Abstract
Importance of the fieldDevices for the reliable detection of panels of biomarker proteins
facilitated by magnetic bead-based technologies have the potential to greatly improve future
cancer diagnostics. The reason for this review is to highlight promising research on emerging
procedures for protein capture, transport and detection featuring magnetic particles.
Areas covered in this reviewThe review covers applications of magnetic particles in
protein immunoassays in emerging research and commercial methods, and stresses multiplexed
protein assays for reliable future cancer diagnostics. Research literature over the past dozen years
has been surveyed and specific examples are presented in detail.
Expert OpinionMagnetic particles are important components of emerging protein detection
systems. They need to be integrated into simple inexpensive systems for accurate, sensitive
detection of fully validated panels of biomarker proteins to be widely useful in clinical cancer
diagnostics.
Keywords
magnetic particles; proteins; cancer biomarkers; off-line protein capture; cancer diagnostics
1. Introduction
The US National Institutes of Health defines biomarkers as molecules that can be

objectively measured and evaluated as indicators of normal or disease processes and
pharmacologic responses to therapeutic intervention [1,2]. Serum protein levels offer the
possibility of snapshots for health monitoring. Their high potential as measurable
biomarkers for cancer detection has created considerable excitement in the biomedical and
clinical research communities [38]. Certain proteins are overexpressed and secreted into
the bloodstream beginning very early in cancer development. These proteins are often
specific to one or several types of cancer, and levels of panels of such proteins can give a
clear indication of a patients status. Thus, serum levels of proteins can serve as biomarkers
to detect the onset of cancer, as well as to guide therapy.
Single biomarker proteins, such as prostate specific antigen (PSA) used as a prostate cancer
biomarker, typically have insufficient positive predictive value, e. g. about 70% for PSA [9].
Corresponding Author: [email protected], Tel: +860-487-0468, Fax: +806-487-2981.

Declaration of Interest: The authors were supported by a PHS grant from NIEHS/NIH and by the NSF.
Mani et al.
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There is concern that detection of an indolent form of prostate cancer that is undifferentiated
by the PSA test from more serious types leads to unnecessary treatment. Failure to
distinguish between indolent and more aggressive forms of cancer is also a common
problem with other clinically used single biomarkers, including cancer antigen 125 for
ovarian cancer, carbohydrate antigen 199 for pancreatic cancer, and carcinoembryonic
antigen for colon cancer [8]. Thus, panels of cancer biomarker proteins will almost certainly
be more valuable for reliable cancer detection and therapeutic monitoring, and this has been
demonstrated experimentally [3,4,7,10,11].
Currently several biosensor technologies are employed as diagnostic tools for protein
detection [12]. Most technologies employ nanomaterials such as gold nanoparticles,
quantum dots, carbon nanotubes and magnetic particles to improve detection sensitivity
[13]. Low detection limits achieved using nanomaterials can facilitate early cancer detection
and accurate prognosis. Virtually any protein detection method that is sensitive enough and
can analyze very small samples can be used to measure cancer biomarker proteins in serum,
and there is no standard approach at present. For this reason, this article includes any method
that could potentially be used for the detection of cancer biomarker proteins.
Clinical or point-of-care (POC) detection of panels of proteins is a formidable bioanalytical

challenge. For clinical use, detection must be sensitive, multiplexed, accurate, and
reasonably priced. POC requirements are more stringent, requiring fast, automated sample
preparation and low cost, technically simple assay devices. These requirements have not
been fully met by any specific methodology available to clinicians at present. Ideally, the
device should be able to accurately measure both normal and elevated serum levels of
proteins. Concentrations that need to be measured may be in sub-pg mL1 to high ng mL1
ranges for different biomarker proteins in the same sample. In addition, potential interferants
include thousands of proteins present in serum, some well above ng mL1 levels [4,7].
Accurate devices that can achieve all goals are needed to provide high quality data for
biomarker panel validation [14,15] before these protein panels can expected to achieve their
full diagnostic potential.
A method for determining multiple proteins in the same sample must be able to selectively
fish out a set of low concentration protein analytes from a sea containing thousands of other
proteins. It must then be able to selectively differentiate the individual analyte proteins and
measure them with accuracy and high sensitivity. Magnetic beads conjugated with
antibodies or other protein capture agents provide a simple but effective way to achieve
these analytical operations. Typically, labeled magnetic beads with antibodies attached are
added to a fluid sample, and the sample is agitated so that specific proteins are captured by
antibodies on the relevant bead. Magnetic separation, either manual or automated, is used to
remove the protein-laden beads from the sample, and wash away any interfering
biomolecules. Labels associated with the magnetic beads are then detected in a selective
way, either by using different labels for different proteins in a bar-code like approach, or by
first sorting beads with the same labels based on the proteins they have captured, then
detecting labels on each type of bead.
Magnetic beads have high surface areas per unit volume, good stability, and enable fast
kinetic processes involving solution species compared to bulk solid surfaces [16,17]. A great
advantage of magnetic beads, as opposed to non-magnetic nanoparticles, is their ease of
manipulation with simple, inexpensive magnets. Very efficient isolation of analyte proteins
from biomedical samples can be achieved outside of the detection system, so that detectors
need never be exposed to the complex biological sample matrix.
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Magnetic beads enable magnetic purification and enrichment of proteins from complex
serum samples. For example, magnetic beads coated with antibodies are effectively utilized
in quantification of biomarker candidates in plasma at ng mL-1 levels by mass spectrometry
(MS) [18]. This approach enabled identification of protein biomarker candidates for head
and neck cancer [19], autistic spectrum disorder [20] and other diseases. High throughput
lectin coated magnetic particles were used to isolate and analyze glycosylated proteins
utilizing LC/MS for biomarker discovery[21].
The present article describes the utility of magnetic beads in a variety of methods developed
to achieve sensitive protein detection. Magnetic particle-enabled protein assays constitutes a
subset of the larger research area of interfacing nanoparticles or microparticles with protein
bioanalyses [22]. In the next section, we discuss sources and properties of magnetic beads.
We then present sections addressing uses of magnetic beads interfaced with different types
of detectors. Closing sections offer a summary and our opinions.
2. Sources and Types of Magnetic Beads
Many types of magnetic beads are now commercially available. Paramagnetic beads are the
most useful for systems requiring magnetic separation and transport as they become
magnetic in an applied magnetic field, but have zero magnetization in the absence of a
magnetic field. These beads are often called superparamagnetic [23]. Ferromagnetic beads
feature permanent magnetism.
The most common examples of paramagnetic beads have magnetic iron oxide cores and
non-magnetic polymer shells featuring surface chemical functionality for attachment of
biomolecules (Figure 1). The magnetic core can also consist of a collection of paramagnetic
nanoparticles embedded in a polymer core. Beads with sizes in the range 100 nm to 50 m
in diameter are commercially available with variability in size <5 %. Suppliers include
Solulink, Invitrogen, Bangs Labs, Merck, and others. Bead size determines sedimentation
rate and mobility in solution. The outer polymer shell serves to add surface functional
groups to the bead and protects the metal oxide core from external media. The outer shell
can also consist of agarose, cellulose, porous glass or silica. Surface functional groups
include carboxyl, amine, epoxy, hydroxyl, tosyl, and N-hydroxy succinate (NHS)-activated.
Beads are also available with surface molecules such as streptavidin, biotin, protein A,
protein G, IgG, IgE and IgM (Figure 1). Functional groups can be activated for coupling
using the familiar EDC-coupling chemistry for carboxylates or glutaraldehyde for amines to
attach to appropriate functional group of biomolecules [7]. Surface tosyl-, NHS-activated
and epoxide groups can be used to attach biomolecules directly without cross-linking agents.
Particles precoated with streptavidin can capture biotin labeled biomolecules. Protein A
coated particles can selectively bind to Fc regions of antibodies for orientated
immobilization.
Beads with paramagnetic nanoparticles embedded in a polymer core matrix are often labeled
superparamagnetic, but may feature multidomain magnetic structures with remnant
magnetic moments [23]. This can cause some degree of magnetic clustering due to induced
magnetism in neighboring particles. At room temperature, true paramagnetic beads of iron
oxide need to have radii in the low nm range. Thus, commonly used beads of 0.13 m
diameter featuring polymer-embedded iron oxide nanoparticle cores may show some
clustering in dispersions due to magnetic interactions between particles.
In the next section, we present methods utilizing magnetic bead-based assays for enhanced
protein detection (Figure 2) that may eventually be suitable for cancer diagnostics.
Specifically emphasis is on high sensitivity detection of multiple proteins, and methods
include magnetic field sensors, electrochemistry, surface plasmon resonance,
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electrochemiluminescence (ECL), polymerase chain reaction (PCR) and bio-barcode assays

achieved using magnetic particles as platforms or labels. Table 1 summarizes selected
results from these methods.
3. Magnetic particles as platforms

3.1 Electrochemical detection
Electrochemical sensors employing capture antibodies and enzyme-labeled secondary
antibodies in sandwich immunoassays can provide high sensitivity, selectivity, low cost, and
instrumental simplicity [7,24,25]. Coupling electrochemical sensors with magnetic beads
hold significant promise for clinical diagnostic devices. Magnetic beads have been used in
enzyme-linked immunosorbent assay (ELISA) formats or as labels for protein detection
[67,2632]. Magnetic beads conjugated with primary antibodies can be held onto a sensor
surface by a magnetic field. Target antigens can then be captured onto the magnetic beads
followed by adding enzyme-labeled secondary antibodies. A current proportional to protein
concentration can be generated by injecting a solution containing a substrate for the enzyme
label and using a potential to electrolyze the enzyme product, or by using a mediator that
exchanges electrons with the enzyme.
Willner and Katz summarized the use of magnetic beads for controlling magneto-switchable
bioelectrocatalytic processes applicable to immunosensors [33]. For example, they reported
enhanced bioelectrocatalytic oxidation of glucose by soluble glucose oxidase by using
ferrocene-functionalized magnetic particles on a rotating magnetic electrode, where
ferrocene acts as the mediator [34]. Wang et al demonstrated ultrasensitive detection using
anti-IgG coated magnetic beads that captured IgG followed by addition of DNA/anti-IgGfunctionalized polystyrene beads to form a sandwich immunocomplex [35]. Oligonucleotide
labels that corresponded to IgG concentration were released in alkaline solution, depurinated
by acid, and the free guanine released was measured by stripping potentiometry. Using
similar immunoassays, carbon nanotubes coated with thousands of alkaline phosphatase
enzymes were employed to achieve an ultralow detection of 67 aM IgG using square wave
voltammetry [36]. Tang et al. developed an electrochemical magnetic-controlled
microfluidic device for the multiplexed detection of 4 tumor markers, -fetoprotein (AFP),
carcinoembryonic antigen (CEA), cancer antigen 125, cancer antigen 15-3 with DLs < 0.5
ng mL1 [30]. Sarkar et al reported electrochemical detection of f-PSA using magnetic
beads on a screen-printed sensor [37]. Magnetic bead protein capture was done in a cuvette,
then, the beads were transferred to the sensor surface. The amperometric response of HRP
labels on the bead was developed using hydrogen peroxide to give a DL < 0.1 ng mL1 fPSA.
Liu et al. reported a multianalyte electrochemical immunoassay involving dual binding

events, based on different semiconductor labels linked to different antibodies and magnetic
bead coated antibodies [38]. Zinc sulfide, cadmium sulfide, lead sulfide and copper sulfide
nanocrystals were conjugated with anti-2-microglobulin, anti-IgG, anti-bovine serum
albumin, and anti-C-reactive protein antibodies, respectively, to bind with their target
proteins on the magnetic bead. The nanocrystals were dissolved in acid and resulting metal
ions were detected with stripping voltammetry to achieve fM detection limits (DL). Zani et
al demonstrated an electrochemical PSA immunoassay using protein G modified magnetic
beads with 8-electrode screen printed arrays [39]. After PSA was captured on magnetic
beads equipped with alkaline phosphatase enzyme labeled antibodies, the product of enzyme
conversion of 1-naphthyl-phosphate was detected by differential pulse voltammetry for a DL
of 1.4 ng mL1. Selvaraju et al. [40] reported an electrochemical immunoassay for mouse
IgG using antibody modified magnetic beads and gold nanocatalysts (AuN) with ultra low
detection limit of 100 ag mL1(0.7 aM) by differential pulse voltammetry. Immunocomplex
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in which the target protein is captured by both antibody modified magnetic beads and AuNconjugated antibody was attracted onto ferrocenyl-tethered dendrimer (Fc-D) modified
indium tin oxide electrode using an external magnet. AuN in the immunocomplex produces
p-aminophenol from the catalytic reduction of p-nitrophenol in the presence of NaBH4. The
p-aminophenol is electrochemically oxidized to p-quinone imine via electron mediation by
ferrocene, and p-quinone imine is then reduced back to p-aminophenol by NaBH4. The
redox cycle amplifies the signal.
Aptamers are short sequences of nucleotides designed to be specific for target proteins, and
have also been used as capture agents and labels for sensitive protein detection. Aptamer
based assays include magnetic particles as solid supports, and polymerase chain reaction
(PCR), electrochemical and fluorescence for aptamer label detection. Electrochemical
aptamer based sandwich assays have been developed to detect C-reactive protein (CRP)
[41], thrombin and activated protein C (APC) [42].
3.2 Optical detection: Electrochemiluminescence
Electrochemiluminescence (ECL) detection involves electrochemically generated light

emission from a luminescent label, and is also promising for multiple protein assays. The
most frequently used label is tris (2,2-bipyridyl) ruthenium(II), [RuBPY], which initiates
ECL when its oxidized form reacts with a suitable sacrificial reductant such as
tripropylamine [43]. Visible light emission occurs upon electrochemical driving of this
process, thus simplifying instrumentation compared to other luminescence approaches. A
typical ECL magnetic bead format features antibody-streptavidin-magnetic beads that
capture protein analyte. RuBPY-labeled biotinylated secondary antibody is added to bind to
the streptavidin, and the beads are washed and magnetically captured onto an electrode for
ECL measurement [43].
Magnetic bead methods similar to that describe above are the basis of protein detection
instruments such as Roches ELECYS, Igens Origen and BioVeriss M-Series. Commercial
measurement systems and kits are available for up to 10 proteins. Fluid handling systems
assist transport of magnetic bead and ECL labels to the detecting electrodes in 96-well
plates. Measured ECL light intensity corresponds to the amount of protein present in the
complex medium. Commercial systems have typical DLs of 110 pg mL1 for most
proteins, and usually require a non-reusable 96-well plate for each assay. ECL with RuBPY
labels has been used for the detection of cancer biomarkers proteins such as
adrenocorticotropic hormone [44], cardiac troponin T [45], parathyroid hormone (PTH)
[46], C-reactive protein [47,48] and alpha-fetoprotein (AFP) [49]. ECL using 100 nm
RuBPY-silica nanoparticle labels has been used for the detection of PSA in cancer patient
serum [50].
3.3 Magneto-PCR based assay
Wacker et al. reported a magneto-IPCR (immuno-PCR) utilizing a sandwich
immunocomplex formed on magnetic particles with antibody conjugated DNA fragment as
labels. The labels are detected using real time PCR to obtain the protein concentration.
Magneto-IPCR was used to detect hepatitis B surface antigen (HBsAg) with a detection
limit of 320 pg mL-1 [51].
Csordas et al. reported a micromagnetic aptamer PCR (MAP) technology to detect protein
biomarkers in serum samples [52]. This technique utilized antibody-coated magnetic
particles which capture biomarker proteins in serum followed by binding of a specific
aptamer. Then, this complex was magnetically separated and the aptamer was amplified
using PCR to provide high sensitivity detection. Platelet-derived growth factor-BB (PDGF-
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BB) over the range 62 fM to 1 nM was detected using MAP technology in complex serum
samples. In a related approach, Tennico et al. used sandwich assays to detect thrombin
utilizing aptamer coated magnetic beads, quantum dot labels, and fluorescence microscopy
[53].
4. Magnetic particles as labels

4.1 Magnetic Field Sensors
Since biological applications of giant magnetoresistance (GMR) sensors were first reported
over a decade ago [54], several magnetic field protein biosensors [5558] with magnetic
bead labels have been described. Detection has been done by magnetic field sensors
including GMRs, superconducting quantum interference devices (SQUID) [59] and Hall
effect [60]. GMR detects magnetic bead labels by measuring the resistance change of a
conducting sensor featuring alternate layers of ferromagnetic and non-magnetic materials in
nm thicknesses under applied current and magnetic field. In a typical example, the
conducting sensor layer has attached primary antibodies to capture the protein of interest
from the sample. After protein capture in this so called sandwich immunoassay, the
surface is washed and a biotin-labeled secondary antibody is added to bind to the target
protein. This is followed by incubation with streptavidin-coated superparamagnetic bead
labels that bind strongly to biotin on the secondary antibody (Figure 3). Detection of
proteins is achieved by measuring the change in resistance of the conducting layer due to the
superparamagnetic bead labels. This method provides high sensitivity at low sensor cost and
low background noise.
Palma et al. used streptavidin coated magnetic beads in a magnetic immunosensor to detect
stroke and minor head injury marker S100 with a detection limit of 27 pg mL1[61]. The
Naval Research Laboratory (NRL) developed a GMR sensor chip array with 64 sensors
called the bead ARray Counter (BARC) measuring biomolecules by detecting
superparamagnetic labels [54]. Mulvaney et al. reported magneto-electronic based detection
of proteins in complex matrices utilizing magnetic bead labels and fluidic flow
discrimination (FFD) [62]. Beads captured on the sensor surface were measured using
microscopy and BARC sensors. FFD lowered the non-specific binding and increase the
sensitivity of detection for proteins. A 64 sensor-GMR array [63,64] enabled multiplexed
detection of proteins biomarkers in clinical samples utilizing magnetic nanoparticle labels
with sensitivities in femtomolar and attomolar ranges. Eight protein biomarkers associated
with various cancers were detected using this sensor, with insensitivity to matrix effects. The
small size and portability of GMR sensor arrays have fueled interest in this approach for
protein biosensing at point of care [65].
4.2 Surface Plasmon Resonance

Surface Plasmon resonance (SPR) is an evanescent wave optical reflectance method
sensitive to refractive index (RI) differences in surface films up to 300 nm thick above a
gold sensor surface. In SPR immunoassays, for example, binding of a protein to a surface
antibody changes the RI by increasing the thickness of the surface film. In a typical
sandwich immunoassay, the sensor is coated with primary antibody and captures the target
protein. If a secondary antibody labeled with a magnetic particle then binds to the captured
analyte, a larger RI increase and larger signal will result due to the size and higher RI of the
magnetic particle.
This approach has been used in SPR-based immunoassays for high sensitivity protein
detection [6668] Magnetic beads can provide off-line target capture and magnetic
separation to decrease non-specific binding of interferants in the sample (Figure 4).
Teramura et al. reported SPR immunoassay for proteins using biotin labeled secondary
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antibodies (Ab2) captured by streptavidin coated magnetic nanobeads to detect brain

natriuretic peptide (BNP) with a DL 25 pg mL1 in buffer [66]. Soelberg et al. used 50 nm
streptavidin-coated magnetic nanobeads as labels to detect staphylococcal enterotoxin B
(SEB) using SPR at DL 100 pg mL1 in stool samples [67]. Krishnan et al. recently utilized
1 m superparamagnetic magnetic bead (MP)-Ab2 bioconjugates for off-line antigen capture
of prostate cancer biomarker PSA in serum. These particles were then captured on a primary
antibody coated SPR biosensor and an ultralow DL of 10 fg mL1 (300 aM) [68]. Detection
was greatly amplified by utilizing aggregation of the MP-Ab2 on the SPR sensor surface
(Figure 5), which provided high sensitivity via a very large RI change per bound protein.
Accuracy of SPR detection in this assay was validated using cancer patient serum.
4.3 Electrochemical detection
Nanostructured amperometric immunosensors were used for PSA in serum employing HRPlabeled magnetic beads with DL of 0.5 pg mL1 [69]. Tang et al. reported signal
amplification using thionine doped magnetic gold nanospheres and HRP to detect
carcinoembryonic antigen [70] at 5 pg mL1. Sandwich immunoassay was fabricated on
carbon fiber microelectrode modified with anti-CEA/protein A/nanogold particles.
Electrochemical signals were developed by the reduction of H2O2 by labeled gold
nanospheres. Ambrosi et al. reported magnetic microbead based electrochemical
immunoassay utilizing gold nanoparticle labels for signal enhancement with a detection
limit of 260 pg mL1 of human IgG [71].
4.4 Other detection method: Biobarcode assays
Magnetic beads have been used extensively as solid supports to covalently link
biomolecules such as antibodies, DNA and enzymes. Antibody coated magnetic beads
provide stronger binding for target proteins by using mechanical agitation. Ultrasensitive
detection of several proteins in serum has been achieved using biobarcode-based detection
utilizing magnetic beads as solid supports [6]. In this approach, gold or silica nanoparticles
act as carriers for polynucleotide strands of known-sequence that serve as biobarcodes. The
barcode is the sequence of nucleobases in the polynucleotide label that can be read by a
complimentary polynucleotide strand. Nam et al. used primary antibody coated magnetic
beads to capture target antigen followed by washing and then binding of secondary
antibody-gold nanoparticles [72] or silica microparticles [73] with the biobarcodes attached.
After magnetic separation of the immunocomplex, the biobarcodes are released by using
high temperature and low salt concentration, and detected using a scanometric or
colorimetric assay. The polymerase chain reaction (PCR) can be used to enhance sensitivity
to very high levels. Biobarcode assays can provide attomolar-level detection of protein
biomarkers in serum. Multiplexed detection of prostate specific antigen (PSA), human
chorionic gonadotropin (HCG) and -fetoprotein (AFP) was achieved with this approach
utilizing 3 different antibody coated magnetic beads with detection limits of 170 fM in
diluted serum [74].
5. Summary
Using magnetic particles for protein capture, manipulation, transport, and labeling has led to
methods that detect cancer biomarker proteins at clinically relevant serum levels and below.
GMR and SPR detectors benefit from the inherent signal enhancement of the magnetic
particle itself, which does not need additional labels. Approaches such as biobarcode assays
and nanostructured microfluidic arrays with massively labeled particles can detect proteins
well below pg mL1 levels in serum. Off-line capture of the analyte proteins by magnetic
particles ensures that the detectors need never be exposed to the sample, eliminating many
Mani et al.
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possible interferences and greatly lowering non-specific binding (NSB) which is a major
source of background in immunoassays.
Despite the above successes, only a small fraction of the methods discussed in this article
have been validated using cancer patient samples. However, the few that have been tested
with patient samples have demonstrated good accuracy and sensitivity. In addition, cancer
diagnostics applications using protein biomarkers will require accurate detection of not a
single protein, but panels of 410 biomarkers for each cancer. Much less progress has been
made on this front, with approaches to multiplexed protein detection systems for clinical or
POC use just beginning to appear in research publications. Currently no commercial devices
for multiplexed cancer biomarker protein detection are suitable for POC use. Commercial
magnetic bead-based methods utilizing ECL seem promising for laboratory-based
multiplexed protein determinations, but are relatively expensive and technically demanding.
6. Expert opinion
Clearly, magnetic bead-based technologies are becoming firmly established as important

components of the protein detection systems of the future. However, it will be necessary for
such systems to detect small panels of proteins and other biomarkers to be most useful for
clinical diagnosis. In addition, POC devices will require simple, low cost, technically
undemanding methodology. Extensive testing and validation will be necessary on patient
samples such as serum and saliva to establish analytical reliability, and clinical sensitivity
and selectivity. Interfacing of sensitive, reliable, simple detection protocols with
microfluidic or other automated sample handling technologies seems a necessary future step
to reach POC. However, the payoff is potentially high for future cancer diagnostics and
therapy monitoring. Extensive research and development efforts are underway with the hope
of solving these formidable challenges. An exciting dream for the future is a hand-held
multiplexed protein detection device about the size of a cell phone that can carry out all
sample handling, measurement, and data analysis tasks for panels of biomarker proteins for
a given cancer, or better yet, for 100 or more biomarkers for the most prevalent cancers.
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Article Highlights

Magnetic beads facilitate sensitive methodologies for multiple protein detection

important for future cancer diagnostics.
Magnetic beads with polymer coatings are commercially available in diameters

from 100 nm to 50 m with surface chemistries amenable to attaching virtually
any biomolecule.
Paramagnetic beads are most useful for protein capture, separation, and
transport, but may have remnant magnetism leading to some degree of
aggregation.
Magnetic beads without surface biomolecules can serve as signal amplifying

labels in Surface Plasmon Resonance (SPR) and Giant Magnetoresistance
(GMR) sensors.
Use of multiple labels such as enzymes, polynucleotides, or metal complexes

either attached to magnetic beads or partner nanoparticles can be used to greatly
enhance sensitivity in optical and electrochemical immunoassays.
There is a significant need for validation of magnetic bead-based assays and

biomarker panels with sufficiently large numbers of patient samples.
Inexpensive, technically undemanding, automated protein detection devices will

be required for future point of care cancer diagnostics and therapy monitoring.

Mani et al.
Page 14

Figure 1.
Superparamagnetic beads are commercially available with coatings of either organic

functional groups to attach biomolecules like antibodies and enzymes, or precoated with
biomolecules that can bind specific partners.
Mani et al.
Page 15

Figure 2.
Schematic representation of the use of magnetic particles (MP) as supports (I and III) and
labels (II) for protein biomarker measurements using different detectors.

Mani et al.
Page 16

Figure 3.
Schematic representation of giant magnetoresistance (GMR) biosensor for the detection of

antigen based on the sandwich immunoassay. The sensor detects fringe field of magnetic
bead labels under applied in-plane magnetic field. Scheme is adapted from [54].

Mani et al.
Page 17

Figure 4.
Illustration of off-line capture of protein analytes using labeled magnetic beads, a technique
that can be used with nearly any type of detector. Magnetic beads with bound protein are
selectively captured on the sensor surface, while beads without analyte are washed away.
Magnetic and SPR methods require only the magnetic bead as a label

Mani et al.
Page 18

Figure 5.
Signal amplification due to aggregation of magnetic particles on SPR sensor surface in

immunoassays for the detection of PSA using surface plasmon resonance (SPR) assay.

Mani et al.
Page 19
Table 1
Part A. Protein detection limits for methods using magnetic particles as labels or platforms
Part B. Protein detection limits
Biomarker
Off-line capture?
Detection limits
Coating of particles
References
Magnetic field sensor

No
27 pg mL1
300 nm streptavidin coated
61
Cancer 63CEA), Eotaxin, granulocyte colony

stimulating factor (G-CSF), interleukin-1-alpha
(IL-1), interferon-, lactoferrin, tumor necrosis
factor alpha (TNF-)
No
mL1
1 pg
each
biomarker
63
CEA, VEGF, lactoferrin, survivin, EPCAM, TNF, GCSF, Eotaxin
No
CEA 1pg mL1,

VEGF 10 pg mL1
64
C-reactive protein (CRP)
No
25 ng mL1 in
buffer
0.51 m streptavidin
coated
75
S100 protein
Bio-barcode assay
Prostate specific antigen (PSA), human chorionic

gonadotropin (HCG),-fetoprotein (AFP)
Yes
170 fM in serum
1 m tosyl particles
76
Amyloid--derived diffusible ligands(ADDLs)
Yes
100 aM in CSF
1 m polyamine
76
Interleukin-2 (IL-2)
Yes
30 aM in serum
1 m amine-functionalized
77
Prostate specific antigen (PSA)
Yes
3 aM in buffer
1 m amine-functionalized
72
Surface plasmon resonance assay

Brain natriuretic peptide (BNP)
No
25 pg mL1
50 nm streptavidin
66
Staphylococcal enterotoxin B (SEB)
Yes
100 pg mL1 in
stool
50 nm streptavidin
67
Yes
10 fg mL1 in
serum
1 m tosylated
68
Electrochemical assay
0.83 m streptavidin
36
25 nm silica nickel ferrite
30
< 0.1 ng mL1
3 m tosylated
37
No
0.5 pg mL1 in serum
1 m carboxyl functionalized
69
Yes
fM levels
Streptavidin coated
38
Protein G coated
39
IgG
Yes
Carcinoembryonic antigen (CEA), cancer antigen 125,

cancer antigen 15-3, -fetoprotein (AFP)
No
<0.5 ng
Free (f)-PSA
Yes
PSA
IgG, BSA, 2-microglobulin, C reactive protein
Carcinoembryonic antigen
Human IgG
Mouse IgG
Yes
No
Yes
Yes
67 aM
1.4 ng
mL1
mL1
mL1
20 nm gold nanosphere
70
260 pg
mL1
Streptavidin coated
71
100 ag
mL1
1 m Tosyl activated
40
5 pg
Electrochemical aptamer based assay

C-reactive protein,
Yes
5.4 102 mg/L
1.05 m streptavidin
41
Thrombin
Yes
0.5 nM in buffer
1.05 m streptavidin
29
Activated protein C (APC)
Yes
1 nM
Protein G coated
42
320 pg mL1 in serum
Antibody functionalized magnetosome
51
Magneto-PCR
Hepatitis B surface antigen (HBsAg)
Yes
Mani et al.
Page 20
Platelet-derived growth factor-BB(PDGF-BB)
Yes
1 m streptavidin
52
350 nm streptavidin
78
62 fM to 1 nM serum
Electrochemiluminescence (ECL)
CEA protein
No
1.6 pg mL1
L1
2+
Adrenocorticotropic hormone (ACTCH)
Yes
C-reactive protein (CRP)
Yes
0.01 g mL1 in buffer
1 m streptavidin-
47
Human CRP
Yes
100 ng mL1 in buffer
2.8 m streptavidin-
48
0.5 ng
Ru(bpy)3

ECL label
44

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Published in final edited form as:

Magnetic particles in ultrasensitive biomarker protein

of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut

The US National Institutes of Health defines biomarkers as molecules that can be

Corresponding Author: [email protected], Tel: +860-487-0468, Fax: +806-487-2981.

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Clinical or point-of-care (POC) detection of panels of proteins is a formidable bioanalytical

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2. Sources and Types of Magnetic Beads

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electrochemiluminescence (ECL), polymerase chain reaction (PCR) and bio-barcode assays

3. Magnetic particles as platforms

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Liu et al. reported a multianalyte electrochemical immunoassay involving dual binding

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Electrochemiluminescence (ECL) detection involves electrochemically generated light

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4. Magnetic particles as labels

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4.2 Surface Plasmon Resonance

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antibodies (Ab2) captured by streptavidin coated magnetic nanobeads to detect brain

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Clearly, magnetic bead-based technologies are becoming firmly established as important

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NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

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Magnetic beads facilitate sensitive methodologies for multiple protein detection

Magnetic beads with polymer coatings are commercially available in diameters

Magnetic beads without surface biomolecules can serve as signal amplifying

Use of multiple labels such as enzymes, polynucleotides, or metal complexes

There is a significant need for validation of magnetic bead-based assays and

Inexpensive, technically undemanding, automated protein detection devices will

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

Superparamagnetic beads are commercially available with coatings of either organic

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

Schematic representation of giant magnetoresistance (GMR) biosensor for the detection of

NIH-PA Author Manuscript

NIH-PA Author Manuscript