Advanced Drug Delivery Reviews: Charalambos Kaittanis, Santimukul Santra, J. Manuel Perez

Advanced Drug Delivery Reviews 62 (2010) 408–423
Contents lists available at ScienceDirect
Advanced Drug Delivery Reviews

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r
Emerging nanotechnology-based strategies for the identification of

microbial pathogenesis☆
Charalambos Kaittanis a,b, Santimukul Santra a, J. Manuel Perez a,b,⁎
a
Nanoscience Technology Center, USA
b
Burnett School of Biomedical Sciences – College of Medicine, USA
a r t i c l e i n f o a b s t r a c t
Article history: Infectious diseases are still a major healthcare problem. From food intoxication and contaminated water, to
Received 26 May 2009 hospital-acquired diseases and pandemics, infectious agents cause disease throughout the world. Despite
Accepted 14 September 2009 advancements in pathogens' identification, some of the gold-standard diagnostic methods have limitations,
Available online 13 November 2009
including laborious sample preparation, bulky instrumentation and slow data readout. In addition, new field-
deployable diagnostic modalities are urgently needed in first responder and point-of-care applications. Apart
Keywords:
Pathogen detection
from compact, these sensors must be sensitive, specific, robust and fast, in order to facilitate detection of the
Deployable sensors pathogen even in remote rural areas. Considering these characteristics, researchers have utilized innovative
Toxin detection approaches by employing the unique properties of nanomaterials in order to achieve detection of infectious
Bacterial drug resistance agents, even in complex media like blood. From gold nanoparticles and their plasmonic shifts to iron oxide
Nanosensors nanoparticles and changes in magnetic properties, detection of pathogens, toxins, antigens and nucleic acids
Nanodiagnostics has been achieved with impressive detection thresholds. Additionally, as bacteria become resistant to
antibiotics, nanotechnology has achieved the rapid determination of bacterial drug susceptibility and
resistance using novel methods, such as amperometry and magnetic relaxation. Overall, these promising
results hint to the adoption of nanotechnology-based diagnostics for the diagnosis of infectious diseases in
diverse settings throughout the globe, preventing epidemics and safeguarding human and economic
wellness.
© 2009 Elsevier B.V. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
2. Current technologies for infectious agent diagnosis and their limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
2.1. Isolation, growth and microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
2.2. From microscope-based detection to nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
2.3. Detection of pathogen markers with antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
2.4. Detecting toxins — approaches for a challenging task . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
2.5. Limitation of current technologies — the potential of nanotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
3. Available nanotechnologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
3.1. Gold nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
3.2. Silver nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
3.3. Quantum dots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
3.4. Fluorescent polymeric nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
3.5. Magnetic nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
3.6. Nanochips and nanoarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
3.7. Fiber-optic-based biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
3.8. Cantilever-based arrays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414
4. Approaches for detection of infectious diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414
4.1. Pathogen identification through surface marker recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414
4.2. Pathogen detection using nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Nanotechnology Solutions for Infectious Diseases in Developing Nations”.
⁎ Corresponding author. Department of Chemistry, University of Central Florida, 12424 Research Parkway, Suite 400, Orlando, FL 32826, USA.
E-mail address: [email protected] (J.M. Perez).
0169-409X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2009.11.013
C. Kaittanis et al. / Advanced Drug Delivery Reviews 62 (2010) 408–423 409
4.3. Detecting toxins and infectious diseases secreted markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417

4.4. Determining drug resistance/susceptibility through the monitoring of a pathogen's metabolic activity . . . . . . . . . . . . . . . . 418
5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
additional transmission routes involving mosquitoes, co-habitation in

Nomenclature close contact with infected animals and contaminated water, socioeco-
nomic trends and political instability of several developing nations are
AFM atomic force microscopy additional factors that synergistically contribute to the spread of
Ag NPs silver nanoparticles infectious diseases [4]. Thus, improving the living conditions and
AIDS Acquired Immunodeficiency Syndrome diagnostic protocols in poor rural areas is critical in controlling the
Au NPs gold nanoparticles spread of disease before becoming a worldwide pandemic. Also, as
CTAB cetyl trimethylammonium bromide modern global traveling facilitates the spread of the disease faster than
D-Ala D-alanine-terminated peptidoglycan ever, developing fast, simple and accurate methods to identify infectious
D-Lac D-lactate-terminated peptidoglycan diseases is of timely importance.
DNA deoxyribonucleic acid Infectious diseases are caused by contagious agents (pathogens) that
ELISA enzyme-linked immunosorbent assay are capable of inducing disease with symptoms that can be manifested
FISH fluorescence in situ hybridization within a couple of minutes, or after a couple of hours to days or even
FRET fluorescence resonance energy transfer years after the initial infection. These pathogenic agents are subject to
HAADF-STEM high angle annular dark field scanning transmis- transmission from either an infected individual or vector (such as ticks,
sion electron microscopy birds or pigs) to a healthy individual [4]. The complexity and broad
HIV human immunodeficiency virus range of pathogens that cause disease, in addition to the prolonged
HPLC high performance liquid chromatography incubation time of some of these agents before clinical symptoms of the
Kd equilibrium dissociation constants disease are present, make the diagnosis of some of these conditions even
LC–MS liquid chromatography mass spectrometry more challenging. Pathogens that cause disease can be listed within
MALDI-TOF-MS matrix-assisted laser desorption/ionization- various groups, such as bacteria, viruses, fungi, protozoa, parasitic
time-of-flight mass spectrometry worms, and prions. Their unique characteristics, ways of transmission,
MAP Mycobacterium avium spp. paratuberculosis as well as any associated disease biomarkers, such as toxins, antigens
MDR-TB multidrug-resistant Mycobacterium tuberculosis and nucleic acids are listed in Table 1. The diversity of these pathogens
MRI magnetic resonance imaging resides not only on the nature of the disease they inflict in the host, but
mRNA messenger ribonucleic acid also in their size and shape (see also Scheme 1).
MRSA methicillin-resistant Staphylococcus aureus Nanotechnology presents a great opportunity to develop fast,
MudPIT multidimensional protein identification accurate and cost effective diagnostics for the detection of pathogenic
NIR near infrared infectious agents [5,6]. Due to the presence of unique properties in
OD optical density nanoscale materials, devices able to report the presence of a pathogenic
PCR polymerase chain reaction agent in clinical or environmental samples can be designed. The prop-
PDMS polydimethylsiloxane erties observed in nanomaterials are different from those observed in
Qdots quantum dots the bulk (micron-size) material due to their small size (1–100 nm) and
R2 spin–spin relaxivity large surface area, resulting in enhanced surface reactivity, quantum
RAPID-PCR random-primed polymerase chain reaction confinement effects, enhanced electrical conductivity and enhanced
RNA ribonucleic acid magnetic properties, among others [6]. Most importantly, modifications
rRNA ribosomal ribonucleic acid of the nanostructures' surface can alter dramatically some of their
SQUID superconducting quantum interference device properties [7,8]. Hence, a single binding event can be potentially
STEM scanning transmission electron microscopy recorded. Because of these phenomena, multiple nanostructures have
T2 spin–spin relaxation time been engineered to detect particular molecular targets in biodiagnostic
UV ultraviolet applications, including pathogen detection. This article focuses on
UV–vis ultraviolet–visible reviewing some of the most promising nanotechnologies available or
XDR-TB extremely drug-resistant Mycobacterium tuberculosis under development for the detection of pathogens that cause diseases.
ΔΤ2 change in spin–spin relaxation time
2. Current technologies for infectious agent diagnosis and

their limitations
1. Introduction
2.1. Isolation, growth and microscopy
Infectious diseases cause significant human pathogenesis and
mortality throughout the world, surpassing cardiovascular diseases Traditionally, the presence of most pathogens such as bacteria, fungi,
and cancer [1]. Although affluent developed countries have made great protozoa and worms is determined microscopically, usually after
progress in sanitation and technological advances to identify and control growth in pure culture. Typically, a sample from the infected individual
most infectious diseases, problems remain with food contamination, is taken and observed in the microscope for the presence of the
hospital-acquired pathogens, and sexually transmitted diseases [2,3]. In pathogen. For bacteria or fungi, subsequent confirmation is based on the
poor developing countries and even in rural areas of developed growth patterns in differential media and via additional biochemical
countries, infectious diseases are a major problem mainly because of tests. These methods, although highly specific, have several limitations.
not only poor sanitation but also the lack of efficient technologies to First, microscopy-based methods work with samples containing a high
identify and treat these conditions in a timely manner [2,3]. Furthermore, amount of pathogens. Second, growth pattern methods usually require
410 C. Kaittanis et al. / Advanced Drug Delivery Reviews 62 (2010) 408–423
Table 1
Typical infectious disease agents. Differences in size, morphology, infection mode, pathogenesis mechanisms, clinical symptomology and disease highlight the need for development
of sensitive and specific pathogen identification modalities in diverse settings.
the growth of the pathogen in a particular medium followed by at least a host's immune response affect the microorganism's expression patterns
24-h incubation period to yield results. Third, as some microorganisms at the transcriptional and translational levels. Apart from PCR, several
cannot grow easily in culture, their identification is even more chal- other molecular diagnostic methods have been introduced, such
lenging. These limitations are even more significant in the identification as RAPID-PCR, checkerboard hybridization, ligase chain reaction,
of viruses that due to their small size (aprox. 100 nm) cannot be studied ribotyping using restriction length polymorphisms, and pulsed-field
using conventional optical microscopy and require the use of an electron gel electrophoresis [4]. Despite their distinct advantages, all of these
microscope for their visualization. Finally, the culture and growth of methods require undamaged microbial DNA and have to be performed
viruses in the laboratory require extensive protocols to grow them in a laboratory setting by experienced personnel and expensive
before analysis. instrumentation and reagents. Therefore, the associated cost of these
molecular diagnostic modalities is high enough, prohibiting their wide-
scale use at the points-of-care and in developing nations.
2.2. From microscope-based detection to nucleic acids
With the discovery of the DNA and the development of Polymerase 2.3. Detection of pathogen markers with antibodies
Chain Reaction (PCR), microbiologists adopted a molecular-based
diagnostic frameset in the last quarter of the 20th century. Specifically, In addition to microscopy- or nucleic acid-based techniques, the
instead of hunting for the microorganism as an entity itself, they started identification of a pathogen can be achieved via immunoassays
looking for genes and proteins associated with its virulence and disease targeting specific proteins or carbohydrate moieties unique to the
patterns. Consequently, there was a rise in genome sequencing and the pathogen. Such methods include, agglutination tests, fluorescent im-
deposition of the annotated genome in databases [4]. Based on this munoassays, enzyme-linked immunosorbent assays (ELISA) and West-
information, DNA microarrays have been developed, identifying DNA ern blot analyses [4]. Although these methods are very sensitive, they
segments corresponding to an organism's genome. These methodolo- detect a pathogen's molecular ‘fingerprints’, such as proteins and cell
gies are highly sensitive and selective, achieving detection down to the wall epitopes. Hence, as these assays do not provide any direct
single pathogen. Despite this, the major limitation of the gene chip is information about the presence and metabolic state of a microorganism
that it cannot provide critical information about a pathogen's specific in the sample, the administration of suitable antibiotic or other ther-
RNA (transcriptional) and protein (translational) levels. Furthermore, it apeutic agents cannot be achieved. Additionally, as these methods
cannot address how these parameters are modulated by factors and regularly utilize an antibody for the recognition of bacterial targets,
processes that may alter the microorganism's growth, as well as how the proper handling and storage is needed in order to prevent antibody
Scheme 1. Size distribution of widely used nanosystems in comparison with the most common types of infectious disease agents. A particular nanoparticle (represented by a black
horizontal dashed line) could interact differently with targets of various sizes, such as peptides, toxins, viruses and bacteria (blue, grey, green and orange dashed lines, respectively).
In this particular case, the nanoparticle has a size of 100 nm, whereas virulence factors and disease markers are smaller (e.g. lipids, peptides, DNA, toxins), and pathogens can be of
roughly equal (e.g. most viruses) or bigger size than the nanoparticle (e.g. bacteria, fungi, etc.).
denaturation. Furthermore, these assays are affected by the clinical For instance, toxins, like listeriolysin O from L. monocytogenes, create
sample's nature, because the specimen, for instance blood, often times pores in and disrupt the phospholipid bilayer, causing host cell's lysis
needs to undergo processing before being analyzed. Similarly, flow [15]. Other toxins, such as Shiga toxin, inhibit protein synthesis, which
cytometry [9–11] and phage typing [10] face limitations imposed by the in turn activate apoptotic and necrotic cascades [16,17]. Furthermore,
sample's characteristics, as optically opaque or viscous samples can toxins, like anthrax and botulism, alter the host cell's signal trans-
interfere with the readout, plus the assay's portability is hindered due to duction cascades. These alterations initiate the disease state and,
the bulky instrumentation. Likewise, magnetic microbeads have been ultimately, lead to the host cell's death [18]. Hence through exploi-
used for the magnetic separation and identification of microbes through tation of numerous mechanisms, bacterial toxins affect host cell's
PCR and flow cytometry [11,12], yet these methods require several homeostasis and induce cell death, aiming to the release of nutrients
intermediate steps and have limited capabilities in complex biological needed for microbial survival and colonization. Particularly, intracel-
samples. lular pathogens that have a potent toxin in their virulence repertoire
Pathogenesis caused by intracellular pathogens, such as Mycobac- utilize the toxin as a key exit mechanism, a tool for further infection
terium tuberculosis among others, relies on the survival of the micro- and manifestation within the body.
organism within host cells, such as macrophages, dendritic cells and T Even though toxins are not usually transmitted among infected
helper cells [13]. This mode of infection and the frequent absence of individuals, their devastating effects on tissues and whole organs merit
clinical symptoms hamper these pathogens' detection and prevent the development of sensitive, fast and reliable diagnostics methods and
treatment and containment. Consequently, the physician cannot therapeutics to minimize their harmful effects. Most importantly, toxins
proceed to the assignment of an appropriate treatment course, delaying might remain in environmental, food or clinical samples long after the
the clearance of the pathogen from the body. Current tests cannot detect corresponding pathogens have been dead. Therefore, screening these
intracellular pathogens in biological fluids, such as the blood and lymph samples for the presence of active toxins is highly important to min-
fluid, as most of these tests are based on fluorescence emission in the imize intoxication and economic losses, particularly during a pandemic.
absence of optical interference. Hence, the need for developing optical- Current toxin detection methods utilize antibody–antigen interactions
independent diagnostic modalities is pivotal for the detection of in- in ELISA [19], Western blots [20,21], antibody microarrays [22], surface
tracellular pathogens in body fluids and biopsies, with no or minimal plasmon resonance biosensors [23], and antibody-coated polystyrene
sample preparation steps. Furthermore, these modalities should be able microbeads [24]. These methods are relatively sensitive and have
to facilitate multi-sensing capabilities, detecting not only the microor- multiplexing capabilities, but require homogeneous or purified samples.
ganism per se, but genomic fragments and protein markers. This can Although time-consuming and laborious, these purification procedures
corroborate bacterial identification and prevent misdiagnosis due to are critical for assay sensitivity and specificity, as they minimize back-
cross-reactivity [4], which is common in serological assays. ground noise. A major limitation of these assays is their spectrophoto-
metric or fluorimetric toxin determination mode. Thus, limited
2.4. Detecting toxins — approaches for a challenging task biological and environmental samples can be screened. To circumvent
this obstacle, toxin detection techniques that utilize mass spectrometry,
Toxins are virulence factors that affect the host cell's physiological such as liquid chromatography mass spectrometry (LC–MS) [25] and
processes and plasma membrane integrity, causing cytotoxicity [14]. multidimensional protein identification (MudPIT) [26], have been
developed and achieved high sensitivities. However, the lack of particular nanoparticle would interact differently with targets of
portability and user-friendliness as well as the sophisticated instru- different sizes, resulting in different response patterns that are unique
mentation, prevent the broader use of these diagnostic methods. to the particular type of interaction that the nanoparticle exhibits with
the pathogen.
2.5. Limitation of current technologies — the potential of nanotechnology
3.1. Gold nanoparticles
Conventional molecular diagnostic techniques are widely used in
laboratories throughout the world to identify pathogenic agents with Gold nanoparticles (Au NPs) can be synthesized either in an aqueous
high degree of sensitivity and reproducibility. However, most of these or organic environment [33]. Conventional techniques for aqueous
techniques cannot be utilized in the field (e.g. airports and food synthesis involve the reduction of AuCl3 with trisodium citrate, followed
distribution centers) or in developing countries where resources are by the addition of a capping agent to stabilize the Au NPs by introducing
scarce, because they often require sophisticated, expensive instrumen- adequate electrostatic repulsion between individual particles keeping
tation that needs to be used by trained personnel. Additionally, the high them well dispersed in the medium [33]. Alternatively, organic-based
cost and short shelve half-life of some reagents, such as enzymes and Au NPs synthesis offers excellent control and improved size uniformity
DNA primers, limit the application of most conventional pathogen [33]. Surface chemistries of Au NPs can be controlled by grafting
detection techniques in developing nations. Furthermore, despite their functionalized organic thiol molecules or thiol-containing polymers, as
sensitivity, current technologies, like ELISA and PCR, require extensive the gold surface exerts strong affinity towards sulfhydryl groups leading
sample preparation and have long readout times, which delay prompt to the formation of relatively strong covalent bonds. Hence, further
response and disease containment. Hence, taking advantage of the surface modification can be done simply by using thiolated functional
unique electrical, magnetic, luminescent, and catalytic properties of molecules, facilitating conjugation of various probes, including anti-
nanomaterials, faster, sensitive and more economical diagnostic assays bodies and nucleic acids. Successful formation of Au NPs is associated
can be developed that can assist in the battle against microbial with the presence of a surface plasmon band in the preparation's UV–vis
pathogenesis. Apart from striving for sensitivity and speed, nanotech- absorbance profile. The surface plasmon band arises from the coherent
nologists have geared their efforts towards the development of existence of free electrons in the conduction band, due to the small
nanotechnology-based systems that are affordable, robust and repro- particle size. The band shift depends on the particle size, chemical
ducible, making them suitable for applications even in rural areas of surrounding, adsorbed species on the surface, and dielectric constant
developing nations. For instance, researchers try to formulate cheap [34]. Changes in the local dielectric constant of the nanoparticles by
and stable nanoparticles via novel facile synthetic routes, making absorbed biomolecules or the biomarker-induced agglomeration of the
nanoparticle-based diagnostic assays globally accessible and deliver- nanoparticles cause plasmonic band shifts [34]. Hence, this unique
able. Moreover, using innovative approaches, nanotechnology has the characteristic of gold, as well as silver, nanoparticles allows the use of
potential to build assays that can be performed in opaque media, like the surface plasmon band shifts for several diagnostic applications by
blood and milk, without any sample preparation, providing fast and recording the alterations in the UV–vis absorbance spectrum. Apart
reliable results in simple and user-friendly formats as we describe in the from absorption, gold nanoparticles were used as tags for the detection
following sections. and quantification of numerous targets using fluorescence, Raman
scattering, electrical conductivity, atomic and magnetic force techniques
3. Available nanotechnologies [5,6].
Nanotechnology offers many technological advances for pathogen 3.2. Silver nanoparticles
detection. The use of nanoparticles as labels in conjunction with novel
detection technologies has led to improvements in sensitivity and Silver nanoparticles (Ag NPs) were first used as a new generation of
multiplexing capabilities [5,6]. Metallic nanoparticles composed of antimicrobial agents to prevent infections [35]. Generally, these
gold or silver have many optical and electronic properties, derived nanoparticles can be synthesized using methods yielding spherical,
from their size and composition [27]. When coupled to affinity elongated (rod-shaped) or truncated (triangular) Ag NPs [35]. Initially,
ligands, these nanomaterials have found important applications as silver seeds can be prepared by rapidly injecting 10 mM NaBH4 into an
chemical sensors. For example, gold nanoparticles conjugated with aqueous solution of 10 mM AgNO3 and 1 mM sodium citrate and aged
specific oligonucleotides can sense complementary DNA strands, for 1.5 h [35]. Spherical silver nanoparticles can be prepared by
detectable by color changes [7,8]. Other nanoparticles including reducing an aqueous solution of AgNO3 (1 mM) by adding silver seed
fluorescent quantum dots and carbon nanotubes have been used in solution and aqueous solution of sodium citrate, under boiling
various applications including DNA detection, and the development of conditions until the color of the solution becomes greenish-yellow
immunoassays for the detection of bacteria and toxins [28–32]. [35]. Similarly, the elongated and triangular shaped silver nanoparti-
The properties of the nanomaterials used for pathogen detection can cles can be prepared by adding silver seed, 0.1 M ascorbic acid, 0.1 M
be tailored by changing the size, shape, composition and surface CTAB and 1 M NaOH solution to the aqueous solution of AgNO3
modification of the nanomaterial. Particularly, their electronic, spectro- (10 mM) to accelerate particle growth [34,36]. The in situ reduction of
scopic (emissive, absorptive), light scattering and conductive properties Ag+ ions and introduction of surface functional groups can be achieved
can be modified by engineering the nanoparticles' structural parameters, in one-pot reactions, using various biopolymers. Other than small
including their size, composition, self assembly and binding properties reducing agents, such as sodium citrate and hydrazine hydrate, various
[6]. In addition, recent years have seen an explosion in the development functional polymers, including poly(acrylonitrile), poly(N-vinyl pyr-
of surface patterning techniques that promise on generating nanoscale rolidone), poly(vinyl alcohol), polyacrylic acid and polyacrylamide,
arrays of pathogen targeting ligands. These developments could rev- were used for the reduction and surface coatings of the silver ions [37].
olutionize the way how one can detect pathogens and infectious Hence, these functional Ag NPs can perform conjugation chemistries to
diseases. Below, some of the most commonly used nanosystems for the connect various targeting ligands, such as proteins, antibodies,
detection of pathogens are described. It should be noted that a particular peptides, oligonucleotides and small molecules. Ag NPs exhibit a
nanosystem, for example a nanoparticle of 100 nm in size including its surface plasmon band, usually at 422 nm for spherical nanoparticles,
coating and targeting ligands, can be designed to sense targets that might which can be used for colorimetric detection and quantification of
be smaller (i.e. lipids), bigger (i.e. bacteria) or of equal size (viruses) to targets. Also, these nanoparticles can be used for the microscopic
the nanoparticle (Scheme 1). Therefore, it should be expected that this identification of targets using either high angle annular dark field
(HAADF) or scanning transmission electron microscopy (STEM), and is coated with polymers, such as dextran, polyacrylic acid and silica.
electrochemical approaches. Modulation of the nanoparticles' shape and magnetic properties can
be engineered via different strategies, including the time of addition
3.3. Quantum dots of the polymer, higher temperature and the use of particular capping
agents [50]. Further surface modification can facilitate the addition of
Fluorescent quantum dots (Qdots) are semiconductors with unique functional groups, such as amino and carboxylic acids, making sub-
optical properties when compared to conventional semiconductors, sequent conjugations easy. Hence, iron oxide nanoparticles can carry
organic fluorescent dyes and proteins [6]. Qdots can be prepared using a diverse ligands, such as peptides, small molecules, proteins, anti-
variety of methods ranging from molecular beam epitaxy to electron bodies and nucleic acids. Therefore, iron oxide nanoparticles have
beam lithography, yet colloidal synthesis is the most common technique been used for the identification and quantification of several targets,
for Qdots preparation. Specifically, in the case of hot solution-phase including mRNA, DNA, viruses, bacteria and cells [50–54]. Further-
synthesis, periodic group II and VI (CdSe, CdTe, ZnSe, etc) or III and V more, enzymatic and metabolic activities were monitored with these
elements (InP, InAs, etc), are dissolved in a solution with a stabilizing nanoparticles [55–57]. Detection with magnetic nanoparticles can be
agent or polymer. Thus during the synthesis procedure, these salts enter achieved with the use of magnetometers or superconducting quan-
into a phase where they associate forming core-shell Qdots capped with tum interference device (SQUID), which record alterations in the
either a polymer or other stabilizer [38]. Furthermore, a simple ‘cap magnetic properties of the particles upon molecular interactions
exchange’ approach can be used to anchor for instance bifunctional with a target [58–60]. Also, detection and quantification can be
mono- or di-thiols to the Qdot surface, whereas the other functional accomplished with magnetic relaxometers and magnetic resonance
end-group remains available for further functionalization and conjuga- imaging (MRI), by monitoring the changes in the spin–spin relax-
tion chemistries. Mechanistically, it is the band-gap that determines in ation time (T2) of the solution's water protons due to nanoparticle
which frequencies the Qdot will respond to. Hence, by engineering their association with a target [50–54].
band-gap (such as by changing their size), Qdots can emit light in
different wavelengths upon excitation, having broad and diverse
applications. For instance, simply by modulating their size, Qdots can 3.6. Nanochips and nanoarrays
be excited at a given wavelength and have tunable emission from the
ultraviolet (UV) to the near infrared (NIR) region. Apart from being Nanochips can be prepared for the fast identification of biomole-
tunable, Qdots are highly bright and extremely photostable, making cules, including toxins and antibodies, using gold or silica nanoparticles
them suitable for various biomedical applications such as sensing supported on thin silicon layers [5,6]. Following sample incubation, the
and detection of biomarkers including antigens and pathogens, nanochips are subjected to different procedures in order to achieve
immunolabeling of cells and tissues, detection of cancer in vitro and in signal amplification. Due to their small size and use of currently
vivo [28,39–42]. Qdot-based FISH (fluorescence in situ hybridization) available instrumentation, nanochips can screen samples in a high-
probes have been widely used for the detection of the Y-chromosome throughput format requiring minute sample volumes [5,61]. Further-
in fixed human sperm cells [43]. Furthermore, Qdot-FRET-based more, apart from quantifying a target, nanochips can identify if proteins
nanosensors mediated the ultrasensitive detection of low concentra- undergo post-translational modifications, such as proteolytic cleavages
tions of target DNA in the diagnosis of genetic diseases [44,45]. and phosphorylation, using matrix-assisted laser desorption/ionization-
time-of-flight mass spectrometry (MALDI-TOF-MS) [5].
3.4. Fluorescent polymeric nanoparticles In addition to nanochips, nanoarrays have been used to further
expand the emerging field of DNA arrays [5,6,62]. Through different
Polymeric nanoparticles are easily prepared using a linear or manufacturing approaches, nanoarrays have been fabricated using
branched polymer that promotes the encapsulation of a fluorophore inkjet printing, electrochemistry, microfabricated surface patterning,
within the nanoparticle's cavity or hydrophobic microdomains [46–48]. or photolithography with either pre-assembled mask or micromirror-
Several synthetic routes can be utilized yielding stable monodispersed based apparatus [5]. Specifically, the nanoarray can be constructed on
nanoparticles in aqueous media. The resulting nanoparticles, such as diverse surfaces, such as silicon, silanes, hydrogels, metallic and
fluorescent silica ones, can be filtered and concentrated. As the presence polydimethylsiloxane (PDMS), among others. Hence, detection of a
of the polymeric coating protects the fluorophore, these nanoparticles target can be achieved with AFM, surface plasmon resonance, optical
are stable under diverse conditions [46–48], surpassing organic dyes in waveguides, nanowires, microfluidics and electrical current detectors.
photostability and versatility. Additionally the presence of functional The nanoarray can utilize various probes, including antibody, nucleic
groups on the polymer coating, including amines, carboxylic acids and acid and small molecule, which can be either deposited in 1–30 µm
esters, confers facile attachment of probes. Surface functionalization and droplets or via fluidics-enhanced molecular transfer operation [62]. In
targeting can be achieved via numerous conjugation chemistries, such addition to these entities, colloids and Qdots can be further applied
as “click” and carbodiimide [49]. Detection is mainly achieved with the leading to the construction of multiplexed nanoarrays capable of
use of a fluorescence spectrometer, flow cytometer, and fluorescence- screening minute samples in large libraries of potential pathogenic
recording microtiter plate reader [46–48]. agents or therapeutics.
3.5. Magnetic nanoparticles

3.7. Fiber-optic-based biosensors
Magnetic nanoparticles have been used for a long time in the
clinic and the molecular biology laboratories [5,6]. For instance, super- For the rapid detection of analytes, researchers have modified
paramagnetic iron oxide nanoparticles, composed of a magnetite/ traditional ELISA plate assays, developing sandwich-format fiber-
maghemite core, have been utilized as contrast agents for magnetic optic-based platforms [63–65]. Immobilization of a capturing moiety
resonance imaging (MRI). Also, magnetic nanoparticles conjugated (antibody, aptamer, and ligand) to a fiber optic or capillary tube
to antibodies have been used for the immunomagnetic separation of mediates the capturing of the molecule of interest, whereas a
nucleic acids, proteins, viruses, bacteria and cells [5,6]. Iron oxide secondary probe (i.e. antibody) conjugated to a fluorophore facilitates
nanoparticles are primarily synthesized via water-based protocols, target detection. Following fluorophore excitation, the emitted light is
involving the alkaline precipitation of iron salts. To enhance water recorded by portable instrumentation, which can quantify the target
stability and add surface functionality, often times the iron oxide core based on the fluorescence intensity [65].
3.8. Cantilever-based arrays identical travel times, as the photons' point of origin is common [67].
Hence, this approach does not require the removal of unbound
Due to their high sensitivity, cantilevers can be used for the detection nanoparticles as these nanoparticles have a discrete state, which can
of molecular targets. These cantilevers resemble atomic force micros- be easily differentiated from that of the nanoparticles interacting with
copy (AFM) tips and can be decorated with nucleic acids, small a target. Also, there is no need to conjugate an antibody on a solid
molecules and antibodies [6]. Upon binding of a target, the cantilever surface, providing faster detection kinetics and good assay sensitivity.
deflects a couple of nanometers, facilitating detection. Since the deflec- Despite its novelty and sensitivity, its' clinical and field applicability
tion is proportional to the amount of target binding, cantilever-based may be limited as this method utilizes a microcapillary flow system
arrays are quantitative [6]. Advantages of these arrays include the and a fixed-point confocal microscope. In other reports, researchers
absence of any enzymatic amplification steps such as PCR, the detection using a single quantum dot construct have achieved the high-
of nucleic acids and proteins without the need of labels (i.e. throughput quantification of human T cell leukemia virions, demon-
fluorophores), and the ability to be used in microfluidic setups. strating the ability of quantum dots to detect targets via readily
available fluorescence microtiter readers [68]. Thus, one would
4. Approaches for detection of infectious diseases anticipate the use of such diagnostic modalities in the field detection
of viruses and in the determination of novel antiviral inhibitors.
4.1. Pathogen identification through surface marker recognition Recently, quantum dot barcodes have been used for the detection of
viruses, such as HIV and Hepatitis, via a sensitive handheld diagnostic
Pathogens express different markers on their surface to serve a system [69].
variety of purposes. Receptors, glycoproteins, glycopeptides, lipopro- A major challenge for field diagnostics that employ antibodies as
teins, carbohydrates, and lipids participate in host infection, adhesion, targeting ligand is the need to maintain the antibody's structure and
immune system evasion, nutrient uptake and transport, among other prevent thermal denaturation. Considering this challenge, gold
activities. Despite their wide repertoire of functions, these entities nanoparticles and fluorescent π-conjugated polymer constructs have
share a common feature; that is, they are exposed to the extracellular been used for the fluorescent-based identification of microorganisms
milieu. Hence, these biomolecules are attractive pathogen association without the need of antibodies [70]. In this technique, the electrostatic
markers in nanotechnology, as nanoprobes can easily access them interaction between cationic gold nanoparticles and anionic polymers
without having to cross any biological membranes or barriers, such as led to fluorescence quenching [70] (Scheme 2). However, in the
the thick peptidoglycan layer. Utilizing this fundamental property, presence of bacteria, the negatively charged bacterial cell wall caused
nature and the immune system have evolved clearance mechanisms displacement of the nanoparticles' polymer [70] (Scheme 2). Hence,
to recognize these surface markers through highly specific antibody– the interaction between the nanoparticles and bacteria and the
antigen associations. Acknowledging the high specificity of antibodies concomitant dissociation of the polymer from the nanoparticles
towards microorganisms' surface epitopes, nanotechnology has induced the release of the polymer's quenched fluorescence, leading
utilized antibodies as targeting ligands in a wide array of diagnostic to enhanced fluorescence emission (Scheme 2). A library of three
modalities. nanoparticle preparations was prepared and distinct fluorescence
For the fluorescent-based quantification of bacteria, Tan et al. used emission patterns were observed for each organism, including E. coli,
antibody-conjugated dye-doped silica nanoparticles [46,47]. After a B. subtilis, L. lactis and S. coelicolor [70]. Subsequent quantitative
brief incubation with the specimen, any unbound nanoparticles were analysis for pattern recognition through linear discriminant analysis
removed through short centrifugation rounds, and single bacterium led to the construction of a signature plot, having each microorga-
detection was achieved in less than 20 min [46]. Apart from detecting nism's characteristic fluorescence emission [70]. Consequently, this
a single E. coli O157:H7 in processed ground beef samples, other approach can be utilized for the affordable and robust identification of
bacterial species were quantified, such as Salmonella and Bacillus, microorganisms without the need for heat labile antibody-conjugated
indicating that this method can be used for both Gram-negative and probes. It should be noted that as this method is at its infancy, the
Gram-positive microorganisms [46]. Although, both the fluorescent detection threshold was high (OD600 = 1; 1 × 109 colony forming
nanoparticle quantification method and the gold-standard method of units). However, with further optimization and the use of more
plate-counting yielded similar results, the nanoparticle assay's responsive polymeric conjugates that employ a diversified association
readout time was faster than that of the plate-counting (16–18 h). with the nanoparticles, a higher sensitivity should be achieved.
Also, an advantage of the nanoparticle method is its' high-throughput Although pathogen detection has been predominantly achieved
capability using a microtiter plate reader, which can achieve bacterial with optical methods, alternative strategies have been sought to
quantification from 1 to 400 bacterial cells. Thus, its clinical utility is detect targets in opaque media and complex matrices. For example, as
significant, especially for the detection of highly infectious agents few as 25 Salmonella enterica bacteria were detected using silicon
where exposure to low concentrations may be lethal. nitride cantilevers [71]. In this case, bacterial detection was achieved
Viruses have also been identified using fluorescent nanoparticles. by monitoring the cantilever's surface bending, which was directly
Specifically, europium(III)-chelate-doped nanoparticles were able to associated to the amount of bacteria associating on the cantilever [71].
quantify as little as 5000 virions per mL via a sandwich-based immu- Other researchers have achieved quick and specific bacterial identi-
noassay, utilizing microtiter plate-conjugated capturing antibodies fication using an assay that relies on the specific interaction between
and nanoparticle-bound detecting antibodies [66]. Furthermore, using bacteriophage and bacteria. Particularly, it utilizes the bacteriophage's
this method the presence of adenovirus was quantified in nasopha- ability to infect certain bacteria and not others. Thus bacterial lysis
ryngeal patient samples, demonstrating that fluorescence is not leads to changes in the sample solution's composition, which can be
compromised in slightly heterogeneous matrices [66]. In an interest- assessed electrochemically through variations in the electric field of
ing approach, Nie et al. have achieved the detection of the respiratory nanowell apparatuses [72].
syncytial virus via dual-color emitting quantum dots and fluorescence Lastly, among the most innovative pathogen detection methods are
energy transfer nanobeads [67]. Contrary to other approaches, this those that employ magnetic nanoparticles. In early studies, antibody-
design relies on the principle of discrete flight times of photons after carrying magnetic nanoparticles have been used for the detection of a
excitation of the nanoparticles (Qdots and nanobeads) by a single target being immobilized on a mylar film, via superconducting
source [67]. Specifically, nanoparticles that do not associate with a quantum interference device (SQUID) [58]. While any free nanopar-
target emit photons that reach the detector at different times [67]. ticles quickly relaxed by Brownian motion thus not affecting the signal,
However, nanoparticles that bind to a target emit photons that have the nanoparticles that bind to the target undergoes Néel relaxation
Scheme 2. Fluorescence-based detection of bacteria using cationic gold nanoparticles and anionic fluorescent π-conjugated polymers. In the presence of the bacterial anionic cell
wall, there is displacement of the polymer leading to fluorescence emission. Discrete fluorescence emission patterns corresponding to different microorganisms can be obtained in a
high-throughput format. Adapted from reference [70].
leading to a gradually dissipating magnetic flux that the SQUID could the nanoparticles switch to a quasi-dispersed-like state, which
detect [58]. Thus based on this principle, the food-borne pathogen L. resembles the nanoparticles in sterile control [51]. Hence, at high
monocytogenes has been quantified with a detection limit of 6 × 106 bacterial concentrations the T2 is proximal to that of the negative
bacteria [59]. Further improvements in SQUID-based detection have
been accomplished with the development of immunomagnetic
reduction assays [60]. Briefly, these assays' underlying principle relies
on the magnetic nanoparticles' differential oscillations in the presence
or absence of a target during exposure to ac magnetic fields [60].
Interestingly, in the presence of the avian flu virus the anti-H5N1
magnetic nanoparticles clustered. This specific interaction affected the
nanoparticles oscillatory mode, as compared to those of the
corresponding negative controls [60]. Additionally, the oscillatory
pattern obeyed a target-concentration-dependent pattern, facilitating
sensitive H5N1 quantification [60].
Perez et al. have utilized iron oxide nanoparticles that behave as
magnetic relaxation switches altering the spin–spin relaxation times
(ΔΤ2) of adjacent water molecules, upon target recognition [53,54].
With the aid of either a benchtop magnetic relaxometer operating at
0.47 T (20 MHz) or an MRI at 1.5 T (60 MHz), antibody-carrying
dextran-coated iron oxide nanoparticles were able to quickly detect as
little as 5 viral particles of Herpes Simplex or Adenovirus in 10 µL 25%
serum samples [54]. However, the sensitivity was slightly affected
when detection was performed in 100% serum samples, where the
detection threshold was 10 virions per 10 µL [54]. Other molecular
targets such as DNA/RNA, proteins, and enzymatic activities (pro-
teases, telomerase and myeloperoxidase) have been detected using
this technology. However, all these targets are smaller or of equal size
(in the case of a virus) to the nanoparticles used (Scheme 3).
Surprisingly, in the presence of targets larger than the nanoparticles,
such as bacteria, an interesting phenomenon occurs [51]. Initially, in
the absence of any bacteria the nanoparticles are in a dispersed state
(Scheme 3). However, addition of even a few bacteria induces the Scheme 3. Detection of viruses and bacteria using magnetic relaxation. An increase in
assembly of the nanoparticles on the bacterial surface [51]. This the concentration of the virus facilitates clustering of the nanoparticles and high
interaction induces prominent changes in the T2, as compared to the changes in the spin–spin relaxation times (ΔΤ2). On the other hand, at low
concentrations of bacteria high ΔΤ2 are obtained due to nanoparticle assembly on
corresponding T2 values of the sterile control, allowing bacterial the bacteria surface. ΔΤ2 decreases as the bacterial concentration increases because the
quantification at very low concentrations (low colony forming units) nanosensors switch to a dispersed-like state, reminiscent of the one observed in the
(Scheme 3) [51]. On the other hand, at higher bacterial concentrations, sterile medium. Adapted from references [54] and [51].
control (sterile medium) (Scheme 3) [51]. Accordingly, utiliz- HIV sequence [73]. In this case, nominal diffraction response was
ing spherical iron oxide nanoparticles, Mycobacterium avium spp. observed, which was 25 times lower than that of the target sequence
paratuberculosis (MAP) was quantified in whole milk and blood within [73]. Considering its sensitivity, specificity, portability, and simplicity,
less than 30 min [51]. In the absence of bacterial interference caused by this method may be ideal for point-of-care diagnostics. Furthermore,
the presence of several other bacterial species (106 colony forming other researchers utilized gold nanoparticles for the colorimetric
units), roughly 16 MAP colony forming units were quantified, where- detection of DNA-binding molecules, allowing the optical identification
as under interference conditions the detection threshold was of molecules binding to DNA via the solution's color changes.
compromised (∼ 39 MAP colony forming units) [51]. Notably, Collectively, naked eye profiling of pathogens and identification of
detection and quantification of this bacterium particularly at low effective antimicrobial DNA/RNA-intercalating agents should be feasible
concentrations was achieved in blood [51]. Furthermore, this single- with these nanosystems. In line with this, researchers developed a
step nanoparticle assay was able to determine if blood samples from an colorimetric “spot-and-read” assay for the detection of the mecA gene,
individual were MAP positive or negative [51]. This demonstrates the which is found in methicillin-resistant strains of S. aureus [74]. Upon
direct clinical utility of this assay which can expedite diagnosis due to hybridization of the gold nanoparticles with the target DNA sequence
its specificity and elimination of laborious sample preparation and spotting on an illuminated glass waveguide, visual changes were
procedures. Considering these findings, it has been hypothesized observed, leading to bacterial DNA quantification in the zeptomole
that nanoparticles with higher R2 relaxivity may be more sensitive range (zM = 10− 21 M) [74]. However, although a highly innovative
probes for pathogen identification in complex media, achieving technique, as with all nucleic acid-based methods, this assay requires
bacterial detection and lower detection thresholds [50,51]. Most bacterial isolation, lysis and isolation of bacterial DNA which can limit its
recently, dextran-coated iron oxide nanorods with high relaxivity application in field diagnostic and in developing countries.
(300 mM− 1 s− 1) were synthesized and used for the detection of MAP, Another pathogen-associated nucleic acid detection method utilizes
achieving a detection limit of 6 MAP colony forming units in whole the gold nanorods' second-order nonlinear properties [75,76]. Specifi-
milk within 5 min [50]. cally, researchers were able to quantify HIV-1 DNA with high specificity
In similar studies, vancomycin-conjugated iron oxide nanoparticles using hyper-Rayleigh scattering spectroscopy that monitors changes in
were able to quantify S. aureus with comparable to the aforementioned the light's parameters upon target recognition [75]. Simply, using gold
anti-MAP nanosensors' sensitivities, using a handheld diagnostic mag- functionalized nanorods and without any modification or enzymatic
netic resonance system [52]. Interestingly, instead of an antibody, the amplification, researchers have detected 100 pM of the HIV-1 gag
nanoprobe targetability was mediated via a vancomycin–peptidoglycan gene, which encodes one of the viral structural proteins (p55) [75].
bacterial cell wall interaction [52]. Overall, these data demonstrate that Interestingly, this approach was very selective, as single base-pair
the magnetic relaxation switches can achieve enhanced sensitivity by mismatches in the target sequence affected the hyper-Rayleigh
engineering their relaxivity and geometry, using currently available scattering [75]. Additionally, the signal intensity from the complemen-
instrumentation such as compact relaxometers [51,52], without the need tary target sequence can be increased up to 45 times with a slight
for MR imagers as the measurement is one-dimensional and tomographic modification of the system, drastically improving the assay's detection
analysis is not required. Lastly, magnetic relaxation switches can be threshold [75]. This may be particularly important for the identification
adopted in chip biosensor formats to mediate the multiplexed identifi- of highly contagious pathogens or scarce microorganisms isolated from
cation of infectious agents in complex media with portable magnetic various samples. Furthermore, gold nanorods and hyper-Rayleigh
relaxivity devices, suitable for routine and first response screening under scattering facilitated the detection of Hepatitis C virus, a single-stranded
diverse settings and conditions. RNA virus, without performing any amplification [76]. Using this
technique, 80 pM of Hepatitis C viral RNA was detected with single
4.2. Pathogen detection using nucleic acids nucleotide selectivity [76]. Hence, apart from viral RNA, potentially
pathogen-related messenger and ribosomal RNAs can be detected, with
In nature, differences among organisms arise from their variations applications in the clinic and the pharmaceutical industry, as single point
at the genomic level, or differential alterations in the expression of mutations may completely abrogate a drug's antimicrobial efficacy.
their genes and protein modifications. Scientists have sequenced the As most conventional nucleic acid detection methods achieve their
genome of numerous pathogens, identifying unique nucleic acid sensitivity through a polymerase-mediated amplification, research
signatures that are not present in the human genome. Acknowledging efforts have been geared towards the achievement of higher sensitivities
the numerous advantages of nanotechnology, nanoparticle conjugates in nanotechnology-based assays through either short PCR rounds or
of nucleic acids have been designed as probes for the fast iden- assay-intrinsic signal enhancement. For instance, after performing PCR
tification of several pathogens. amplification on clinical samples for various rRNA genes, Sanguinetti
Early reports by Mirkin et al. have demonstrated the unique in- et al. have identified the presence of various Mycobacteria, using a
teraction between gold nanoparticles and DNA, leading to distinct shifts Nanochip microelectronic array and fluorophore-conjugated species-
in the gold nanoparticles' surface plasmon resonance peak [7,8]. Apart specific DNA oligonucleotide probes [77]. In a high-throughput format,
from fast and highly specific, the single-strand-DNA-carrying nanopar- the probes were able to identify the presence of bacteria such as M.
ticles facilitated the real-time DNA detection with micropatterned gold avium, M. xenopi, M. intracellulare, and M. chelonae, whereas nominal
diffraction gratings and gold nanoparticles [73]. Both the diffraction signal, comparable to the negative control, was observed in samples
grating and the nanoparticles had thiolated oligonucleotides as probes, containing other pathogens [77]. Hence, this method may be beneficial
which were complementary to non-overlapping sequences of the for the identification of complex diseases, involving various microorgan-
anthrax lethal factor DNA sequence [73]. Therefore, hybridization isms or microbiota shifts. Instead of using PCR, Wang et al. have
between the complementary target sequence and the probes facilitated developed a visual gene-detecting technique for Hepatitis B and C
the association of the target sequence and the gold nanoparticles with viruses through a sandwich hybridization assay that uses gold
the diffraction grating, causing changes in the local dielectric environ- nanoparticles and silver staining [78]. Serum samples from patients
ment that subsequently affected the incident light's diffracted pattern and control individuals were screened on a Hepatitis B, Hepatitis C, or a
[73]. As this pattern is directly associated with alterations in the local dual (Hepatitis B and C) gene chip that had directly immobilized
microenvironment, various target DNA concentrations were quantified, oligonucleotides and supplemented oligonucleotide-conjugated gold
with an estimated detection limit of 40 fM [73]. Corroboration of the nanoparticles, serving as capturing and detection probes respectively
assay's specificity was achieved by examining the diffraction pattern [78]. As this method is fast and cheap, and requires no sophisticated
upon incubation of the nanoparticles and the diffraction grating with an instrumentation for readout, rural clinics and response teams may
benefit from this technology. Likewise, the attractive strategy by Nam et intoxicated products, such as milk, spinach, nuts, and meats, has
al. was devised for the detection of anthrax lethal factor DNA with caused significant cases of intoxication, even in countries with
remarkable sensitivity (500 zM) and single nucleotide specificity stringent product handling regulations and vigilant public health
[79,80]. The assay's sensitivity relied on an elegant intrinsic signal monitoring agencies. Hence, this indicates that current toxin identi-
enhancement mechanism, utilizing iron oxide microparticles and gold fication methods have limitations, including their complexity, high
nanoparticles [79,80]. Specifically, the magnetic iron oxide microparti- cost, and limited point-of-care utility. Considering these, and the
cles carried a short stretch DNA probe, whereas the gold nanoparticles possible use of toxins as bioterrorism agents, nanotechnology has
carried a different short DNA probe and a DNA duplex, which served as provided us with a plethora of toxin detection approaches, achieving
the amplifier (Scheme 4) [79,80]. The two particle preparations were low detection and portability, while maintaining low cost and user-
incubated with the isolated target DNA, followed by magnetic separation friendly setups.
and DNA duplex dehybridization via heating at 60 °C [79,80]. The latter Among the most prominent and affordable nanotechnology-based
step resulted in the release of the non-conjugated strand (barcode) of toxin detection venues are colorimetric assays that utilize nanopar-
the DNA duplex, facilitating signal enhancement, as multiple bar-code ticle probes. For instance, using thiolated lactose derivatives and gold
strands were released per target recognition event (Scheme 4) [79,80]. nanoparticles, researchers were able to detect cholera toxin through
Despite its' impressive sensitivity, as the nanoparticle-barcode detection molecular mimicry, as the nanoparticles' coating resembled the
requires a couple of hours for data readout and multiple isolation steps, extracellular matrix terminal portion of GM1 ganglioside which is
its' field utility may be limited, yet its' laboratory applicability should be found in the apical membrane of intestinal epithelial cells [81]. The
foreseen as this method is robust, cheap and does not use protein-based detection and quantification were achieved visually and spectropho-
enzymes. tometrically, through color changes in the nanoparticle suspension
(red to deep purple) and shifts in the nanoparticles' plasmonic band,
4.3. Detecting toxins and infectious diseases secreted markers as increases in the toxin concentration facilitated concomitant red
shifts in the UV–visible absorption spectra [81]. The assay provided
As toxins are potent biomolecules causing pathogenesis in a wide- results within 10 min, having a detection limit of 54 nM (3 µg/mL)
range of populations, quick identification of these agents is critical. and not being susceptible to interference caused by ions and proteins
Within the last years, frequent recalls of produce at a global level [81]. Similarly, by modifying the nanoparticle probing moiety and
occurred, resulting in severe economic losses and damaging interna- molecular mimicry, gold nanoparticles conjugated to globotriose
tional trade relations. Additionally, the delayed identification of were able to detect the Shiga-like toxin, as the toxins B subunit
Scheme 4. Barcode-based detection of B. anthracis DNA using magnetic microparticles (MP) and gold nanoparticles (GNP). After magnetic isolation and DNA dehybridization, release
of the barcode DNA occurs. The barcode DNA can be separated using magnetic isolation and can be detected using a chip-based setup that utilizes gold nanoparticles and silver
staining enhancement. Adapted from references [79] and [80].
specifically interacted with the nanoparticles' carbohydrate moieties biotin moieties that anchored to the surface-interacting streptavidin
that mimicked Gb3 (globotriaosylceramide) found on intestinal molecules. Although not as sensitive as other methods, ricin con-
microvilli and renal epithelia [82]. Because Shiga-like toxin intoxica- centrations above 10 ng/mL (0.2 pM) were detected, suggesting that
tions by bacterial strains, such as E. coli O157:H7, cause bloody further improvements in the device architecture and signal amplifi-
diarrhea and in severe cases hemolytic uremic syndrome that may cation may significantly improve this multiplexing-capable system
lead to death, researchers have engineered carbohydrate-carrying [90].
glycopolydiacetylene nanoparticles for the rapid and selective Apart from detecting toxins, it is often desirable to identify secreted
quantification of this toxin [83]. Similar to the gold nanoparticles, markers, such as antigens and antibodies produced by the host.
recognition of the toxin by the nanoparticles resulted in changes in Especially, the latter is of major clinical significance, mainly in the
the nanoparticles' absorption profile, thus making these nanoparticles cases of intracellular pathogens and asymptomatic carriers who may
attractive agents in biorecognition assays [83]. participate in the transmission of a disease without knowing that they
Since portability is a key element for the detection of toxins, have been infected with an infectious agent. Nanotechnology has
antibody-carrying gold nanoparticles have been immobilized to addressed antigen and antibody detection by employing a diverse
immunochromatography strips and allowed the visual detection of repertoire of methodologies, ranging from barcode amplification to
aflatoxins in grain samples [84]. This assay's detection limit was immunochromatography, and from colorimetry to magnetic relaxation.
0.5 ng/mL, and results were obtained in just 15 min, matching HPLC For instance, for the battle against HIV/AIDS, researchers have managed
analysis data [84]. Therefore, as no sophisticated equipment is needed to detect 0.1 pg/mL of the p24 capsid antigen via nanoparticle-based
and results are obtained visually, farmers and health officials may use barcode amplification [91]. This method in addition to being highly
the gold-nanoparticle-based test strip method for the rapid identifi- specific and yielding no false-positive results, it was more sensitive than
cation of toxins. In addition to their use as direct colorimetric probes, ELISA and was able to detect HIV infection 3 days before ELISA could do
nanoparticles have been used as capturing entities to mediate the so [91]. Considering that the nanoparticle-barcode assays was used for
isolation and sensitive detection of toxins. Iron oxide nanoparticles the detection of amyloid β, an Alzheimer's disease hallmark, in cerebral
conjugated to aflatoxin M1 antibodies were able to magnetically fluid [92], it is envisioned that prions could be detected with this
isolate the toxin from milk, allowing the toxin's quantification in method, potentially preventing the spread of related spongiform
standard ELISA formats [85]. encephalopathies and the annihilation of presumably infected cattle
Striving for multiplexity and higher assay sensitivity, nanoparticle- herds. Furthermore, elegant studies by Rotello et al. have demonstrated
based fluorescent probes have been devised for the detection of that through fluorophore displacement specific proteins can be
toxins. Due to their high fluorescence, quantum dots were conjugated identified, without the need for antibodies [93]. The underlying
to antibodies for the identification and quantification of several toxins principle for this diagnostic method relies on the gold nanoparticle
in sandwich immunoassay formats [31]. Specifically, using a micro- quenching of charge complementary fluorophores, and fluorophore
titer plate reader, cholera toxin, Shiga-like toxin 1, staphylococcal activation upon binding of a target protein to the nanoparticles' terminal
enterotoxin B and ricin were quantified in a high-throughput format groups [93]. Hence, this methodology may be attractive for rural sensing
[31]. Notably, the tunable toxin-detecting quantum dots were able to and developing world applications. Considering these necessities,
determine the exact toxin concentrations in samples that had researchers have utilized immunochromatography and colloidal gold
combinations of these toxins, facilitating simultaneous quantification nanoparticles for the fast point-of-care detection of Herpes Simplex
without the need for multiple isolation and incubation rounds that Virus and anthrax protective antigen antibodies in serum and whole
typical immunoassays require [31]. Interestingly, when europium blood [94,95]. In addition to their robustness and specificity, the
nanoparticles were used in fluorescent immunoassays, detection immunochromatographic test strips were able to detect 3 µg/mL in
sensitivities of 10 pg/mL were achieved [86]. This assay not only serum and 14 µg/mL in whole blood, indicating their potential field
surpasses 100 times in sensitivity the corresponding ELISA ones, but it application [94]. Finally, through magnetic relaxation, Brucella and
had 100% reproducibility versus ELISA's 36% [86]. Furthermore, with influenza antibodies were detected in biological samples, with
these nanoparticles the anthrax toxin protective antigen was detected sensitivities of 0.3 nM and 0.1 pM respectively [96,97].
in serum and blood samples, demonstrating the clinical utility of these
assays [86]. In a different approach, after separation with antibody- 4.4. Determining drug resistance/susceptibility through the monitoring
carrying magnetic nanoparticles, staphylococcal enterotoxin B was of a pathogen's metabolic activity
quantified using fluorescence-based enzymatic nanotransduction
[87]. Through amplification of DNA templates conjugated to anti- An important clinical and public healthcare parameter is the rapid
bodies that recognized the captured toxin, the signal intensity determination of a pathogen's drug resistance. The gold-standard
increased, leading to a detection threshold of 0.11 ng/mL [87]. protocol for the determination of antimicrobial susceptibility relies on
As high sensitivity and selectivity are desired for several clinically the growth of the microorganism in the presence of various drug
important toxins, biosensors are attractive alternatives for lab-based concentrations. In this assay, the appearance of turbidity in bacterial
diagnostics. For instance, gold nanoparticles tethered on a supported cultures in the presence of a drug typically suggests growth and active
GM1-containing lipid bilayer were able to detect the presence of metabolism; hence the drug is either ineffective or is present at a
cholera toxin, via alterations in the diffusion coefficients of the gold concentration that cannot suppress growth. However, lack of turbidity
nanoparticles [88]. Comparing this ligand-based method with indicates that the drug effectively kills the bacteria and concomitantly,
fluorescent immunoassays, the biosensor assay was sensitive more the bacteria are not resistant to that particular drug. Despite its high
than 100 times [88]. Likewise, by employing molecular topology sensitivity and easiness, the turbidity method, similar to other
mimicry, immobilization of lipoic acid-anchored lactosyl and galac- metabolism-based susceptibility assays, provides results at least
tosyl ceramide on a sensor chip via self-assembled monolayer within 24–48 h [98,99].
deposition led to the detection of ricin within 5 min [89]. Due to the Alternatively, most traditional molecular biology-based methods,
surface plasmon resonance analysis's sensitivity, low ricin concentra- as well as contemporary nanotechnological strategies, although fast
tions were quantified (10 pg/mL) [89]. Hence in an attempt to merge and sensitive cannot assess if a pathogen is viable or not, as these
the sensitivity of surface plasmon resonance and the facile construc- methods depend on either immunologic or nucleic acid-mediated
tion of microfluidic devices from polydimethylsiloxane (PDMS), detection, which do not necessarily reflect a pathogen's metabolic
researchers have tethered GM1-containing lipid vesicles on gold state. Therefore, treatment of bacterial infections frequently relies
nanoglassified surface [90]. This was achieved using the vesicles' on empirical associations, sometimes leading to prescription of
ineffective drugs and application of selective pressure mediating drug cannot form hydrogen bonding with the D-Lac peptidoglycans. As little
resistance. Consequently, within the last 20 years there has been an as 10 nM of vancomycin caused significant D-Ala cantilever deflection,
increase in the emergence of antibiotic-resistant microorganisms making this assay clinically relevant, as the typical blood serum
with concomitant elevated pathogenesis at the global level [100,101]. vancomycin concentration is within the range of 3 to 27 µM [103].
Concurrently, there has been a decrease in the development of Most importantly, no D-Lac cantilever deflection was observed in the
antimicrobial agents, due to shifts in the pharmaceutical industry's presence of 7 µM vancomycin in blood, suggesting that this assay can
research and development priorities [1]. Thus, the prevalence of drug- evaluate the interaction between a resistant bacterium and an
resistant strains of Mycobacterium tuberculosis (MDR and XDR-TB) antibiotic at the fundamental single hydrogen bond level, thus expe-
and methicillin-resistant Staphylococcus aureus (MRSA) in the diting the screening of current drugs and the development of novel
developing and developed nations respectively, indicate that drug antimicrobial agents. Interestingly, the vancomycin–mucopeptide
resistance has become a major public health problem [100,101]. An interaction equilibrium dissociation constants (Kd) obtained with the
indication of the problem's magnitude can be obtained through D-Lac and D-Ala cantilevers correlated well with the values obtained
several reports, stating that MRSA infections cause more deaths than through surface plasmon resonance and solution-phase UV spectros-
HIV/AIDS in the United States and bacterial infections are among the copy measurements, indicating that this nanotechnology-based
leading causes of mortality in the industrialized world [100–102]. approach is equally reliable to standard methodologies yet more
Therefore, novel antimicrobial susceptibility systems have to be fast, sensitive (10 nM vs 30 nM detection threshold) [103].
sensitive and cost-efficient in order to assess if a pathogen is resistant A limitation of the cantilever method is that it does not provide any
to first-line antimicrobial agents, and the agents' effective dosage. information about the metabolic state of the microorganism infecting a
Several molecular approaches can be employed for the assessment of patient [3,104,105]. Therefore, a physician cannot assess if an antibiotic
bacterial drug resistance, in addition to the gold-standard metabolism- agents will suppress bacterial growth and viability. Following a different
based turbidity method. For instance, the expression of protein, approach, recent studies reported the assessment of bacterial drug
carbohydrate and glycolipid markers on the bacterial wall, as well as resistance through the monitoring of bacterial metabolic activity, thus
mutations in the genomic level, can facilitate assessment of antimicro- having wider clinical and industrial implications. Among the most
bial resistance. Limitations of contemporary microbial susceptibility interesting nanotechnology-based approaches for assessing the efficacy
assays include extensive sample preparation protocols, long incubation of antibiotics is the use of screen-printed carbon electrode arrays and
times, the use of expensive reagents and limited multiplexed capabil- cyclic voltametry [106]. In these studies, concentrated bacterial
ities. Especially in the developing world, where drug resistance of M. cultures were initially incubated with different antibiotics and
tuberculosis is prevalent, performing these laborious molecular assays is then supplemented with an oxidative solution of ferricyanide and
prohibitory. Thus, clinicians and health care providers rely on culturing dichlorophenolindophenol, followed by amperometric measurements
methods that are simpler and cheaper. However, results are obtained using carbon electrodes [106]. Within less than one hour, the electrodes
after a significant amount of time and in some cases may be inconclusive recorded the amperometric response currents from the reduction of
due to contamination or inadequate sample size. In addition to assay ferricyanide to ferrocyanide, which are markers of electron transfer due
logistics, another critical facet in combating infection diseases in the to bacterial metabolic activity and respiration [106]. Thus, it was
developing world is the identification of the most effective, yet most observed that under inhibitory antibiotic concentrations the ampero-
affordable, drug. Therefore, screening a sample for bacterial susceptible metric currents decreased [106]. This allowed determination of
to multiple antimicrobial agents is too expensive and cumbersome in chloramphenicol's E. coli inhibitory concentration which suppressed
these countries using currently available microbiological methodolo- 50% of the bacterial metabolic activity, potentially facilitating the rapid
gies. This leads to unsuccessful treatment of the infection and potential identification of other effective antimicrobial agents [106]. The use of
development of drug resistance strains of the disease. Hence, developing high concentrations of bacteria (OD ≥ 0.1; 1 × 108 colony forming units),
robust, affordable and faster antimicrobial susceptibility methods can the need for specialized solutions (buffer with growth media and
assist in the administration of effective antibiotics and alleviate the oxidizing cocktail), the absorption of antibiotic by the electrode and the
socioeconomic burden associated with microbial pathogenesis in these non-user-friendly data readout may hinder this method's use in the
countries. field and poor rural areas. However, the pharmaceutical industry
Recently, several steps have been made to this direction, including and government agencies might adopt it due to its sensitivity and
nanotechnological studies that focused on the simulation of the promptness.
interaction between multidrug resistant bacteria and antibiotics using To circumvent these problems, researchers focused on the mon-
cantilever assays and nanomechanical deflections. Specifically, as itoring of nutrient utilization, targeting clinical sample conditions,
vancomycin, a last resort antibiotic for MRSA, interacts with D-alanine- speed and reliability. As previous studies have reported the use of
terminated peptidoglycan moieties (D-Ala mucopeptides) in the carbohydrate-coated gold and iron oxide nanoparticles for the quan-
surface of the bacterium, researchers have developed arrays of silicon tification of glucose via surface plasmon resonance shifts and
cantilevers to assess the formation or absence of interactions between magnetic relaxation respectively [107,108], researchers hypothesized
D-alanine peptidoglycan and vancomycin at clinically relevant con- that these nanoparticles can be used for the quantification of complex
centrations [103]. The aim of this particular work was to study the carbohydrates and the subsequent determination of bacterial meta-
interaction between vancomycin and D-Ala-mucopeptides, ultimately bolic activity in complex media. Recently, Nath and colleagues have
leading to the development of sensitive assays for MRSA detection and synthesized dextran-coated gold nanoparticles, which in the presence
screening arrays for potential drugs that inhibit vancomycin binding to of a carbohydrate-specific clustering-inducing agent (Concanavalin A)
these mucopeptides. Several factors can lead to antibiotic resistance. exhibited starch-concentration-dependent differential clustering
For instance, antibiotic resistance can arise upon subtle changes in the [27]. Notably, at high carbohydrate concentrations extensive cluster-
bacterial cell wall, such as when an amide (D-Ala) group of the bac- ing was observed with concomitant large plasmonic shifts, whereas at
terial cell wall's peptidoglycan moieties is converted into an ester (D- moderate to low concentrations smaller nanoclusters and less prom-
Lac). In these studies, the cantilevers were initially coated with either inent plasmonic shifts were observed (Scheme 5) [27]. Based on these
D-alanine (D-Ala) peptidoglycan from vancomycin-susceptible bacte- findings, E. coli bacteria (1 × 106 colony forming units) were initially
ria or D-lactate (D-Lac) peptidoglycan from vancomycin-resistant incubated for two hours in the presence of various ampicillin con-
bacteria. After incubation with vancomycin, significant cantilever centrations [27]. Subsequently, aliquots of these bacterial cultures
defection was observed in the D-Ala cantilevers. However, minimal were incubated with the dextran-coated gold nanoparticles and
defection was obtained with the D-Lac cantilevers, as vancomycin Concanavalin A, and within an hour they spectrophotometrically
Scheme 5. Determination of antimicrobial susceptibility using gold nanoparticles (GNP). When the levels of complex carbohydrates are high, addition of Concanavalin A facilitates
the extensive clustering of the GNP, with large shifts in the plasmonic band. Alternatively, at low carbohydrate concentrations, the nanoparticles' clustering is less pronounced and
the resulting plasmonic shifts are smaller. Adapted from reference [27].
determined that ampicillin concentrations of 2 µg did not inhibit a result of active bacterial metabolism, should have demonstrated
growth, whereas at 4 µg bacterial metabolic inhibition was achieved larger ΔΤ2, facilitating the determination of antimicrobial susceptibil-
[27]. Furthermore, utilizing these gold nanosensors, Nath et al ity even in opaque media (Scheme 6) [56]. Hence, researchers
determined that the minimum inhibitory concentration for this determined if different microorganisms were resistant or susceptible
bacterial strain was 8 µg of ampicillin, and corroborated these findings to ampicillin. Within 2.5 hours, the dextran-coated iron oxide
with the gold-standard turbidity method [27]. Although the nano- nanosensors determined that E. coli and S. sonnie were susceptible to
particle-based and the turbidity method provided identical results, 8 µg of ampicillin, whereas S. marcescens was resistant to this antibiotic
the nanoparticle-based achieved antimicrobial susceptibility deter- (Scheme 7) [56]. Confirmation of these results was achieved through
mination within an overall readout time of 3 hours versus the the turbidity method after a 24-h long incubation, demonstrating that
turbidity method that required 24 h [27]. Additionally, using a the nanoparticle method is equally sensitive and reliable, yet faster,
microtiter plate reader, a 96-well plate was screened within a couple than this reference procedure (Scheme 7) [56]. Interestingly, Kaittanis
of minutes, facilitating assessment of bacterial drug resistance in a et al. determined these microorganisms' drug susceptibility in blood,
high-throughput format [27]. obtaining identical values with the turbidity method and the magnetic
Acknowledging the need that bacterial drug resistance has to be relaxation method performed in non-blood-containing growth media
achieved in clinical or opaque samples, researchers developed an iron- [56]. Considering these findings, it was reasoned that the direct
oxide-nanoparticle-based assay to facilitate assessment of drug bonding format would provide faster results. In studies with silica-
susceptibility in blood with magnetic relaxation [56]. Two different coated Concanavalin A-carrying iron oxide nanoparticles, it was found
assays have been devised relying on either a competition or direct that these nanoparticles could evaluate bacterial susceptibility with
binding format. In the competition format, dextran-coated iron oxide high sensitivity in just five minutes after addition of the bacterial
nanoparticles competed with the solution's carbohydrates for binding aliquots [56]. Taken together, these findings, as well as those of the
to Concanavalin A [56]. On the other hand, in the direct binding format, carbon electrode array and dextran-coated gold nanoparticles,
Concanavalin A was conjugated to silica-coated iron oxide nanopar- indicate that nanotechnology-based antimicrobial susceptibility
ticles, mediating the nanoparticles' binding to a sample's non-utilized assays can be as sensitive and robust as traditional methods, however
carbohydrates [56]. In initial studies, the iron oxide nanosensors providing faster results which can be useful for the prevention of
facilitated starch quantification and monitoring of bacterial growth epidemics and expedite drug development.
through the changes in the spin–spin relaxation times (ΔT2) [56].
Similar to the dextran-coated gold nanoparticles, it was reasoned that 5. Concluding remarks
samples with inhibitory concentrations would exhibit shifts in the
relaxation times proximal to the sterile medium due to the comparable Nanotechnology is poised to revolutionize the way how pathogen
levels of free complex carbohydrates in solution (Scheme 6) [56]. and infectious diseases diagnostics are performed in the 21st century.
Alternatively, it was hypothesized that samples with non-inhibitory By enabling the fast, cost effective and sensitive detection of infectious
antibiotic concentrations, thus having lower levels of carbohydrates as agents, engineered nanoprobes and nanodevices could facilitate the
Scheme 6. Assessment of bacterial metabolic activity with magnetic relaxation. At small bacterial populations, addition of Concanavalin A induces the formation of large
nanoassemblies of dextran-coated iron oxide nanoparticles (IONP) that have a T2 similar to that of the sterile medium, as the carbohydrate levels are comparable. As the bacterial
population expands and carbohydrates are consumed, smaller nanoassemblies are formed in the presence of Concanavalin A, leading to higher T2 than that of the sterile medium.
Adapted from reference [56].
robust and high-throughput screening of pathogens in biological and are very versatile, because in principle could substitute the use of
environmental samples. This can be achieved via the unique interac- fluorescence molecules as tags in most diagnostics applications. Future
tions these nanoprobes and nanodevices have upon recognition of the trends in nanotechnology will continue in the design of nanostructures
molecular target or pathogen. Inorganic nanoparticles, semiconductor to build miniaturized devices, requiring less sample volumes. In
quantum dots, carbon nanotubes, polymeric nanoparticles, as well as addition, as the field of nanotechnology progresses into the design of
cantilevers and nanochips, all have the potential to be useful in the smart 2-D and 3-D nanoassemblies, new devices that facilitates fast
design of sensitive pathogen diagnostics. Particularly, nanoparticles and sensitive pathogen detection in minimal sample volumes are
Scheme 7. Determination of drug resistance using dextran-coated iron oxide nanoparticles and Concanavalin A. The antibiotic sensitive microorganism (E. coli) had a minimum
inhibitory concentration of 8 µg of ampicillin, which was assessed within 2.5 h with the nanoparticle method. The gold-standard method (turbidity assay) provided results after 24 h.
The nanoparticle method was also able to determine the resistance of a microorganism (S. marcescens) to ampicillin. Adapted from reference [56].
expected. Finally, as drug resistance strains continue to emerge, in the chromatography-digestion-liquid chromatography-electrospray mass spectrom-
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Advanced Drug Delivery Reviews: Charalambos Kaittanis, Santimukul Santra, J. Manuel Perez

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Advanced Drug Delivery Reviews 62 (2010) 408–423

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

Emerging nanotechnology-based strategies for the identiﬁcation of

4.3. Detecting toxins and infectious diseases secreted markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417

additional transmission routes involving mosquitoes, co-habitation in

2. Current technologies for infectious agent diagnosis and

3.5. Magnetic nanoparticles

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