Inorganic Nanoparticles as Donors in Resonance Energy Transfer for Solid-Phase

Bioassays and Biosensors

Yi Han, M. Omair Noor, Abootaleb Sedighi, Uvaraj Uddayasankar, Samer Doughan,

Ulrich J. Krull*

Chemical Sensors Group, Department of Chemical and Physical Sciences, University

of Toronto Mississauga, Mississauga, Ontario, Canada, L5l 1C6

ulrich.krull@utoronto.ca

ABSTRACT

Bioassays for the rapid detection and quantification of specific nucleic acids,

proteins and peptides are fundamental tools in many clinical settings. Traditional

optical emission methods have focused on the use of molecular dyes as labels to

track selective binding interactions, and as probes that are sensitive to

environmental changes. Such dyes can offer good detection limits based on

brightness, but typically have broad emission bands and suffer from time-

dependent photobleaching. Inorganic nanoparticles such as quantum dots and

upconversion nanoparticles are photo-stable over prolonged exposure to excitation

radiation and tend to offer narrow emission bands, providing greater opportunity

for multi-wavelength multiplexing. Importantly, in contrast to molecular dyes,

nanoparticles offer substantial surface area and can serve as platforms to carry a

large number of conjugated molecules. The surface chemistry of inorganic

nanoparticles offers both challenges and opportunities for control of solubility and

functionality for selective molecular interactions by assembly of coatings through

coordination chemistry. This report reviews advances in the compositional design

and methods of conjugation of inorganic quantum dots and upconversion

nanoparticles, and the assembly of combinations of nanoparticles to achieve energy

exchange. The interest is exploration of configurations where the modified

nanoparticles can be immobilized to solid substrates for the development of

bioassays and biosensors that operate by resonance energy transfer (RET).

Introduction

Resonance energy transfer (RET) is a process where energy transfer occurs

between a donor and acceptor via a dipolar coupling and without photon emission

when they are in sufficiently close proximity. The efficiency of this process is

dependent on the distance, degree of spatial alignment of dipoles, and the overlap of

the emission and excitation bands of the donor and acceptor.1-4 The efficiency of RET

can be used for the detection of binding events that bring a donor and acceptor into

close proximity, and has been implemented to determine ensemble and single-

molecule interactions of nucleic acids and proteins. The use of nanoparticle

platforms that can serve as donors has provided opportunity for determination of a

wide variety of molecular interactions with surface-immobilized biomolecules, with

potential for the simultaneous detection of multiple targets by inclusion of multiple

acceptors in the detection strategy.5

Quantum Dot – to - Dye energy transfer

Quantum Dots as donors in Fluorescence Resonance Energy Transfer (FRET)

Several of the unique optical properties of quantum dots (QDs) make them excellent

donors in FRET. These properties include: (i) broad absorption spectra with high

molar extinction coefficient; (ii) narrow and size-tunable photoluminescence (PL)

spectra with high quantum yield (QY), QY of aqueous QDs in the range of 0.2 to 0.7

depending on surface coating6; and (iii) large accessible surface area for

conjugation. A discussion of the details of how these unique optical properties of

QDs influence the efficiency of energy transfer begins with consideration of the

Förster formalism:

E=aR

06

aR06

+r6= a

a+( rR0 )6

(1)

In equation 1, E is FRET efficiency, a is the total number of acceptors that are placed

equidistantly, r, from the same donor and R0 is the Förster distance, which is a

characteristic of a given donor-acceptor FRET pair and is given by Equation 2:

R∘6=8 .79×10−28mol×(n−4 κ2ΦD J ) (2)

The Förster distance, Eq. (2), is characteristic of a specific donor-acceptor pair, and

depends on factors including the refractive index of the surrounding medium, n, the

donor quantum yield, ΦD, the relative orientation between donor emission and

acceptor absorption dipoles, and the degree of spectral resonance between the two

species. These latter two parameters are described by the orientation factor, κ2, and

spectral overlap integral, J, respectively.

J=∫FD( λ )εA ( λ) λ4dλ

∫FD ( λ)dλ

(3)

The spectral overlap integral, Eq. (3), is a function of the fluorescence intensity of the

donor, FD, and molar absorptivity of acceptor, εA, as a function of wavelength, λ,

normalized against the total donor emission. The broad absorption spectra of QDs as

compared to molecular fluorophores allows for selection of an excitation

wavelength where the direct excitation of an acceptor is minimized.3 This ensures

that the majority of the dye acceptors are in the ground state at the excitation

wavelength.1, 3 QDs also have high molar extinction coefficient, which becomes

stronger as the excitation is moved to progressively shorter wavelengths away from

the absorption/excitation spectrum of the acceptor.2 The large “effective” Stokes

shift associated with QDs in combination with strong absorption spectra of QDs

ensures efficient excitation of the QD donor. The broad absorption spectra of QDs

are also useful for the development of multiplexed QD-FRET bioassays, where

multiple colors of QDs can be concurrently and efficiently excited with a single

excitation source.2

The narrow, symmetric and tunable (via size or composition) PL spectra of QDs can

be adjusted for control of the spectral overlap integral in order to maximize the

efficiency of energy transfer without significantly introducing crosstalk between the

QD PL and acceptor emission.3 The near Gaussian PL profile of QDs greatly

facilitates deconvolution of QD PL and acceptor emission from a composite PL

spectrum.1 The relatively high QY of QDs is also useful and must be considered in

the context of the linear dependency of the sixth power of the Förster distance on

the donor QY.1 From the standpoint of multiplexed QD-FRET bioassays, the narrow

and symmetric PL spectra of QDs allows for an integration of a greater number of

color channels within a given spectral window as compared to molecular

fluorophores.1

The large surface area afforded by QDs serves as a scaffold to allow multiple

acceptors, a, to be concurrently arrayed around an exciton donor in a

centrosymmetric configuration, which improves the efficiency of energy transfer.3

This enables acceptors to be FRET paired with a given QD donor despite exhibiting a

weak spectral overlap, owing to the additive channels of energy transfer offered by

multiple acceptors.3 Additionally, arraying of multiple binding sites each with an

acceptor around a single QD donor broadens the dynamic range of QD-FRET

bioassays by extending the quantity of binding chemistry that is available before

saturation is reached.3

The aforementioned discussion assumes that the Förster formalism is applicable to

QDs. Theoretical and experimental studies have confirmed that the Förster dipole-

dipole interaction mechanism applies to QD-dye (donor-acceptor) FRET pairs in

case of direct band gap QDs (e.g. CdSe).7 The inverse 6th power dependence of

energy transfer efficiency on the center-to-center donor-acceptor separation

distance has been confirmed experimentally provided that the donor-acceptor

separation distance is measured from the center of a QD, although there are also

instances where separation distance could be considered from the QD surface.8-10

FRET efficiencies were found to scale with the value of spectral overlap integral, the

number of acceptors interacting with central QD and the donor-acceptor separation

distance that was imposed by the dimensions of a QD.8 The limitation of

approximating a QD as a point dipole is that it places a threshold on the minimum

donor-acceptor separation distance, which is determined by the radius of a QD and

its surface coatings.3 However, this limitation to some extent is mitigated by the

ability to array multiple acceptors around a single QD donor.1 It is also important to

note that the majority of studies that use QD-dye FRET pairs utilize a value of κ2 =

2/3 in the calculation of energy transfer, which is valid provided that the transition

dipoles of donor and acceptor are dynamic and random in terms of orientation.3 For

molecular fluorophores, free rotational motion around single bonds fulfills this

condition despite having a fixed emission dipole orientation. In contrast, CdSe QDs

have been reported to have a degenerate transition dipole that is oriented

isotropically in two dimensions.1 This implies that the assumption of random

orientation of transition dipole is not strictly valid for QDs. Nonetheless, a value of

κ2 = 2/3 is a useful approximation for QD-dye FRET pairs given that multiple

acceptors are arrayed around a central QD over a distribution of positions, where

the acceptors typically have random and dynamic orientation of transition dipoles

with respect to the QD transition dipole and the QD donor has partially random

orientation of transition dipole.3

QDs can serve as FRET donors for development of solution-phase bioassays, and

CdSe/ZnS QDs have been used to develop multiplexed assay strategies for the

detection of nucleic acids. Typically, QDs were conjugated to multiple probe

oligonucleotides and FRET-sensitized emission from molecular dyes that were

associated with complementary target was determined as a quantitative measure of

hybridization. Spectrally resolved simultaneous detection of multiple target

sequences using ensemble measurements and a single excitation wavelength was

possible. Such FRET methodology would have greater practical impact for assay

development if the QDs could be physically immobilized on solid substrates to

develop solid-phase bioassays, and potentially to develop reversible and reusable

biosensors.

Solid substrates as platforms for localization of decorated QDs

Our group has investigated three different types of material for use as physical

supports for decorated nanoparticles in the development of solid-phase QD-FRET

nucleic acid hybridization bioassays. These physical supports include glass and

fused silica substrates in the form of optical fibers11, 12, spherical beads13 and planar

slides14, 15, and also plastic microtiter plates16 and paper substrates17-20. Each of these

solid substrates offers unique advantages and capabilities for the development of

QD-FRET nucleic acid hybridization bioassays.

The popularity of glass and fused silica as substrates is due to the physical

robustness, optical transparency and low autofluorescence of such materials, in

combination with the simplicity and yield of surface derivatization using

commercially available silane coupling agents.21 As examples, optodes that make use

of total internal reflection (TIR) at the sensing region are advantageous for

determination of target compounds at a distance.22 The exponential decay field

associated with an evanescent wave during TIR confines the optical detection zone

to hundreds of nanometers from the surface of an optical fiber, which allows

surface-selective interrogation that can accommodate nanoparticles.21 Glass

surfaces offer stable surface chemistry and good electrical insulating properties

suitable for electrokinetically driven fluid flow within microfluidic channels, which

offers advantages such as small sample volume, higher sensitivity, improved

kinetics for binding interactions such as hybridization, and regeneration of selective

chemistry for multiple cycles of use.23, 24

In contrast with glass and fused silica substrates, plastic microtiter plates are

commonly used in clinical settings for high throughput bioassays.21 Instrumentation

such as microtiter plate readers that are used to interrogate microtiter plates are

widely available in research and clinical laboratories, and such optical readers can

detect FRET emission. Microtiter plates modified with a variety of functional groups

offer opportunity for nanoparticle immobilization, with one example being the

introduction of functional groups on polystyrene surfaces.25 In addition, the

automation provided by robotic sample handling makes microtiter plates appealing

for routine analysis.

Paper-based platforms are attractive for point-of-care and point-of-need diagnostic

applications. Several of the attributes of paper substrates are aligned well with the

ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-

free and deliverable to end users) criteria that have been outlined by the World

Health Organization for implementation of diagnostic technologies in the developing

world and in resource-limited settings.26 Paper is a low-cost substrate based on

polysaccharides, and is commercially available with a variety of different physical

properties such as pore size and flow rates. Paper substrates can be easily patterned

using wax printing to introduce hydrophobic barriers to guide fluid flow and to

fabricate paper-based analytical devices (PADs)27, with capillary action that can

drive fluid flow. Methods for chemical derivatization of cellulosic fibers of paper

substrates are well established, and can be adapted to make use of methods that

were initially intended to achieve immobilization on glass and fused silica.

Additionally, the three dimensional nature of a paper substrate provides improved

cross section of capture (cf. planar glass slide) to enable optical excitation and

imaging using a handheld lamp and a smartphone camera19, respectively, and paper

can be incinerated to eradicate biohazards27. Another significant advantage of a

paper matrix for the development of solid-phase QD-FRET bioassays is the

enhancement of FRET efficiency that has been reported with hydrated28 and dry19

paper formats.

Section 2 expands of chemistries used for immobilization of nanoparticles onto solid

substrates. It is instructive to first consider modification of QD surfaces more

generally, and Section 1 continues with a review of methods that are used to control

aspects such as QD solubility, charge and conjugation.

Decorating Quantum Dots

Quantum dot surface chemistry

QDs with high crystallinity, relatively high QY, monodispersity and narrow PL

spectra are typically synthesized via pyrolysis of organometallic precursors at high

temperatures in organic solvents. As a result, the surface of a QD is capped with

hydrophobic moieties, which is a configuration not suitable for the direct

application of as-synthesized QDs in aqueous biological environments. It is

necessary to modify the surface chemistry of native QDs to impart water solubility.

This is typically done by the addition of a suitable coating to the surface of a QD. The

two commonly reported strategies to make QDs water-soluble are: surface ligand

exchange using thiol or imidazole functionalized small molecules as illustrated in

Figure 1a (i), and polymer encapsulation of QDs as shown in Figure 1a (ii).29

Important considerations for the choice of a method to achieve aqueous solubility of

QDs include: (1) a high affinity by the new ligand for the surface of a QD while

maintaining long-term colloidal stability across a broad range of pH and ionic

strength conditions, (2) subsequent capacity for bioconjugation, (3) maintaining a

compact and small hydrodynamic size of the decorated QD to facilitate the distance

requirements FRET, (4) preservation of optical properties of QDs and functionality

of the attached biomolecule(s), (5) minimization of non-specific adsorption, (6) low-

cost and commercial availability of the QD surface ligand or amenability to a large

scale synthesis of the QD ligand at a low cost, and (7) low toxicity for cellular and in

vivo studies.30, 31

Figure 1. (a) Illustration of two general methods to confer aqueous solubility to QDs.

(i) Ligand exchange of native hydrophobic ligands of QDs by hydrophilic ligands that

coordinate to the surface of a QD, and (ii) polymer encapsulation of QDs where

hydrophobic moieties of an amphiphilic polymer intercalate with the native

hydrophobic ligands of QDs. (b) Chemical structures of selected QD surface ligands.

(i) Schematic of various modules associated with dihydrolipoic acid-polyethylene

glycol (DHLA-PEG) ligand derivatives with various distal functional groups; (ii)

monodentate thioalkyl acid ligands; (iii) bidentate DHLA ligand; (iv) bidentate

DHLA-PEG ligand with different functional groups (R) at the distal end and (v)

tetradentate DHLA-PEG ligand with a methoxy group at the distal end. Panel (a)

adapted with permission from reference30, Copyright 2011 American Chemical

Society. Panel (b) adapted with permission from reference32, Copyright 2014

Elsevier.

In the case of the ligand exchange method, the native hydrophobic surface capping

ligands (e.g. trioctylphosphine (TOP), trioctylphosphine oxide (TOPO) and long-

chain alkylamines) of QDs are replaced with hydrophilic organic molecules that are

typically heterobifunctional.33 The ligand exchange reaction is driven by the high

affinity of hydrophilic ligands that self-assemble via coordination as a monolayer on

the surface of QDs. This process of exchange is also driven by mass action, where

hydrophobic QDs are incubated with a large molar excess of the desired ligand

molecule to thermodynamically and kinetically facilitate the cap exchange.31 QD

surface ligands are comprised of at least two components: proximal anchoring

group(s), and hydrophilic group(s). The anchoring groups are responsible for

interacting with the surface of QDs, while the hydrophilic groups impart solubility in

aqueous media. For many ligands, a distal functional group is also introduced to

provide a site for bioconjugation.

Monodentate thioalkyl acids, such as mercaptoacetic acid (MAA),

mercaptopropionic acid (MPA), and mercaptoundecanoic acid (MUA) shown in

Figure1b (ii), are among the commonly used commercial reagents that can confer

aqueous solubility to QDs. The proximal thiolate group coordinates strongly with

ions such as Cd2+ and Zn2+ present on the surface of QDs (dependent on selected

composition), while the distal carboxylic group when subjected to ionization under

sufficiently basic conditions imparts water solubility and provides colloidal stability

to QDs by electrostatic repulsion.34 While monothiol based coatings tend to be

compact in nature, they suffer from a lack of long-term stability due to high lability

of monothiol ligands at the QD surface.35 A significant shortcoming of QDs capped

with thioalkyl acids is their propensity to undergo aggregation in high ionic strength

or low pH solutions owing to the charge neutralization of carboxylate groups. In

comparison to monothiol ligands, aqueous QDs capped with bidentate thiol ligands

(e.g. dihydrolipoic acid (DHLA) shown in Figure 1b (iii)) offer prolonged shelf life

that ranges from several months to a year.36 This is due to a cooperative chelate

effect of the dithiol functionality as an anchoring group on the same DHLA molecule.

The colloidal stability in case of DHLA-capped QDs is also governed by electrostatic

repulsion between negatively charged carboxylate groups, hence DHLA-capped QDs

are also prone to aggregation depending on pH and ionic strength conditions. 36 To

circumvent this limitation, various modular designs of DHLA-ligand derivatives that

include DHLA appended with polyethylene glycol (PEG) or zwitterionic

functionalities have been used (Figure 1b (i) and 1b (iv)), where colloidal stability in

aqueous conditions is imparted by the interaction of hydrophilic moieties (e.g. PEG

or zwitterionic functional groups) with the solvent instead of reliance on the

deprotonation of distal carboxyl group.35, 36 As a result, these coatings offer colloidal

stability across a wider range of pH and ionic strength conditions, minimal non-

specific adsorption and improved biocompatibility when compared with using only

the DHLA as an anchor.35, 36 Derivatives of DHLA-PEG ligands containing a distal

functional group (e.g. primary amine, carboxyl, methoxy and biotin) have also been

reported, and can be used for bioconjugation and covalent modifications (Figure 1b

(iv).37 In addition to the bidentate DHLA ligands, multidentate (tetradentate) thiol

ligands comprised of two DHLA anchoring groups appended to either a PEG chain or

a zwitterionic functionality can be used (Figure 1b (v)), which further augments

colloidal stability of aqueous dispersions of QDs in a variety of extreme conditions

(pH range 1.1 to 13.9 and 2 M NaCl).38 In contrast with zwitterionic ligands, the PEG-

based ligands exhibit larger hydrodynamic size and can potentially provide a barrier

against metal-affinity driven self-assembly of biomolecules via a histidine (His)

moiety (vide infra).39 An inherent shortcoming of thiols as anchoring groups for QD

functionalization is that thiols are known to serve as traps for holes (electron-hole

pair caused by optical excitation)40, which can greatly reduce the QY of QDs upon

ligand exchange. The holes trapped by thiol ligands on the surface of QDs can also

promote oxidation of thiol ligands to disulfides, which augment the photochemical

instability of aqueous QDs capped with thiol ligands.41 Numerous improvements of

ligand exchange methods have been reported to ameliorate this loss in the QY of QDs

and to augment the colloidal stability of QDs capped with thiol-based ligand. These

improvements include: (1) the use of an organic base to increase the reactivity of the thiol

anchoring group of MPA ligand with the ZnS shell of a QD34, (2) metalation of DHLA

ligand with zinc to produce tetrahedrally coordinated (DHLA)2Zn2 complex which

preserves QD shell structure, i.e., from etching during the cap exchange process42, (3)

using UV irradiation for photochemical transformation of lipoic acid (LA)-modified

ligands to produce heterogenerous population of LA derivatives, where higher order

oligomers (dimers, trimers and tetramers) exhibit faster and stronger coordination for

ZnS-overcoated QDs to promote cap exchange for aqueous solubility43, 44, (4)

ultraefficient cap exchange method requiring ligand-to-QD molar ratio (LQMR) of as

low as 500 (20-200 fold less than most methods), involving the use of tris(2-

carboxylethyl)phosphine for reduction of LA to DHLA and NaOH for deprotonation of

the thiol groups. The low LQMR was beneficial in retaining the original fluorescence of

hydrophobic QDs (>90%) by preventing the QD shell from etching during the cap

exchange process.45 In addition, ligand exchange of hydrophobic ligands with

hydrophilic ligands can also potentially result in unpassivated sites, which promote

quenching of QD PL.30 In addition to thiols, dithiocarbamate ligands derived from

amino acids have also been reported to achieve aqueous solubility of QDs.46

Interestingly, in contrast with bidentate DHLA ligands, QDs capped with

dithiocarbamates exhibit high QY that are similar to values determined for organic

QDs.46

Polymeric encapsulation of QDs has been reported using two different approaches.

Traditionally, this has been accomplished by means of amphiphilic polymers where

pendant alkyl chains intercalate via hydrophobic interactions with native

hydrophobic ligands of QDs (e.g. TOP or TOPO).47 The hydrophilic component of

amphiphilic polymer usually incorporates functional groups (e.g. carboxylic groups

or amines groups) and/or PEG chains to support subsequent chemical modification

and to promote water solubility, respectively. Recently, the hydrophobic portion of

the amphiphilic polymer has also been used to incorporate functional groups for

subsequent immobilization of molecules.48 Polymer encapsulated QDs yield robust

structures with improved optical properties in addition to long-term stability under

a variety of conditions as compared to ligand coated QDs. However, polymer

encapsulation of QDs typically results in a significant increase in the hydrodynamic

size of QDs (> 20 nm)47, which is detrimental for FRET applications owing to the

strong dependency of energy transfer efficiency on the donor–acceptor center-to-

center separation distance. More recently, polymers have been developed which

exhibit coordinating groups such as dithiol49 or pyridine50. These groups interact

directly with the surface of a QD by multidentate ligand exchange interactions,

which serves to reduce the hydrodynamic size of QDs. Additional functional groups,

such as primary amines and carboxylic groups, are also incorporated for further

bioconjugation. In addition to polymer encapsulation of QDs, silica shell

encapsulation of QDs has also been reported where QDs are first ligand exchanged

with a silane coupling agent, such as 3-mercaptopropyltrimethoxysilane, followed

by shell growth that involves hydrolysis and condensation reactions.51 A more

detailed discussion on surface coatings of QDs for aqueous solubility can be found

elsewhere.52

Conjugation of biomolecules to QDs

QDs are made “functional” for use in bioassays and biosensing by conjugation of

biomolecules to the QD surface. The strategies for the preparation of QD-

bioconjugates can be broadly classified into three categories: (1) physisorption, (2)

covalent interaction and (3) coordination linkage.31, 32 Physisorption of biomolecules

on the surface of QDs relies on electrostatic, polar or hydrophobic interaction, and

involves a spontaneous association of biomolecules with the surface coating of a

QD.1 In the case of covalent interaction, a new bond is formed between the

functional group of a biomolecule and a functional group that is associated with the

surface coating of a QD. Coordination linkage is based on dative interaction and

involves spontaneous self-assembly of a functional group of a biomolecule on the

surface of inorganic shell of a QD or the surface coating of a QD.32 Important

considerations for bioconjugation chemistry for the preparation of QD-

bioconjugates include: retention of optical properties of QDs while maintaining

colloidal stability; preservation of the activity of a biomolecule; control of

biomolecule orientation; the stoichiometry of biomolecule conjugation; stability of

the QD-biomolecule conjugate; mild conditions for the preparation of QD-

bioconjugates such that the reaction conditions do not adversely affect the

biomolecule activity, and use of a low concentrations of reactant(s) while

maintaining high yield of biomolecule coupling.

Covalent methods for the preparation of QD-bioconjugates are primarily derived

from chemistries used for protein labeling and make use of functional groups such

as primary amines, carboxyls and thiols.30 These chemistries require additional

reagents for activation such as carbodiimide, succinimidyl ester, maleimide or

pyridyl disulfide. 53, 54 The popularity of these coupling methods arises from

ubiquitous display of carboxyl and amine groups by proteins and that these

functional groups can be easily incorporated into surface coatings of QDs. The

commercial availability of aqueous QDs includes nanoparticles with these functional

groups. However, given the concurrent presence of carboxylic groups and amine

groups on proteins, cross-linking between proteins and formation of QD-protein-QD

constructs are not uncommon from such reactions. This not only leads to mixed

avidity but also results in an uncontrolled variation in the valence of resultant QD-

protein bioconjugates, including limitations in the control of biomolecule

orientation.32 It should be noted that cross-linking is a less severe issue for

oligonucleotide bioconjugation to QDs as oligonucleotides can be easily mono-

functionalized with a reactive group during solid-phase synthesis. In addition to

these chemistries, chemoselective and bioorthogonal methods for the preparation of

QD-bioconjugates that include strain-promoted azide-alkyne cycloaddition,

hydrazone ligation, oxime ligation, alkene-tetrazine ligation and Staudinger ligation

have also been reported.30 The implementation of these chemistries for the

preparation of QD-bioconjugates is described in detail elsewhere.53

Affinity binding based on the avidin-biotin interaction is another coupling chemistry

that has been used for the preparation of QD-bioconjugates. The popularity of

avidin-biotin chemistry originates from its high association constant (Ka) of ca. 1015

M-1, which is one of the strongest known non-covalent interactions.32 The binding is

stable across a wide range of pH and ionic strength conditions. Additionally,

commercial availability of streptavidin (SAv)-coated QDs (SAv is a tetrameric

homologue of avidin that is isolated from Streptomyces avidinii) in combination with

a wide range of commercial kits and reagents for biotinylation makes this

bioconjugation chemistry readily accessible. The biotin functionality can be added

to peptides or oligonucleotides during solid-phase synthesis. Some of the

shortcomings of this coupling chemistry include inability to control the orientation

of biomolecule attachment. In the case of biomolecules that carry multiple biotin

sites, cross-linking resulting in a formation of heterogeneous population of QD-

biomolecule-QD assemblies can be a disadvantage.32 Such cross-linking is not a

major concern for an oligonucleotide strand that is mono-functionalized with a

biotin functionality.

Biomolecules displaying a thiol functional group or a polyhistidine tag can be self-

assembled on the surface of CdSe/ZnS QDs by dative interaction. In the case of a

thiol functionality, the interaction is with the Zn2+ ions or sulfur moieties present on

the QD surface.34, 53, 54 The nature of the QD surface capping ligand responsible for

aqueous dispersion of QDs is a crucial factor in governing this type of interaction, as

the inorganic surface of QD must be accessible. Given the dynamic

association/dissociation of monothiol interaction with QD surfaces, the longevity of

the resulting QD-bioconjugates under non-equilibrium conditions is a significant

concern. The stability of QD-bioconjugates prepared via the dative interaction of a

thiol functionality can be improved by using biomolecules that exhibit multiple

thiols (e.g. dithiol), which improves the stability of linkage by a cooperative

chelation effect.54 However, spontaneous oxidation of a dithiol group to a disulfide

group can impede the stability of resulting bioconjugates. It is also important to

ensure that the thiols that coordinate to the QD surface are not integral to protein

structure and function.

One of the most robust strategies for the preparation of QD-bioconjugates is based

on polyhistidine-metal-affinity interaction, which refers to the ability of histidine

residues to coordinate with transition metals (e.g. Co2+, Cu2+, Ni2+ and Zn2+) via the

imidazole side group that serves as a Lewis base.32 The strong affinity of binding

between a polyhistidine tag and the metal ions can be used for the preparation of

self-assembled QD-bioconjugates. Polyhistidine-metal-affinity driven self-assembly

of biomolecules to the QD surface has been reported using three different methods:

(1) direct coordination of polyhistidine tag to the inorganic surface of QDs; (2)

mutual chelation of Ni2+ ions by carboxylic groups of polymer encapsulated QDs and

a polyhistidine tag associated with a biomolecule; and (3) modification of the QD

surface with nickel-nitrilotriacetic acid (Ni-NTA) groups.32 Each of these approaches

extends the applicability of polyhistidine-metal-affinity interaction for self-assembly

of biomolecules to different QD surface chemistries. Preparation of QD-

bioconjugates using a polyhistidine motif offers a number of advantages. It is a

bioorthogonal means of bioconjugation, as a polyhistidine moiety does not exist

naturally in proteins.32 The single attachment point provides some control of

biomolecule orientation and avoids undesired cross-linking reactions.32 Owing to

the high affinity of binding, there is improved control of the average stoichiometry

of the QD-bioconjugate, ameliorating the need for additional purification steps.32

This method does not compete with hydrolysis, hence facilitates rapid

bioconjugation32 with self-assembly reaching equilibrium within ca. 100-200 s, with

dissociation constants (Kd) in the range of 10-10-10-7 M.39 The relatively small size of

a polyhistidine motif is also beneficial for the retention of native function of a

protein.32

For the case of amphiphilic polymer encapsulated QDs, the inorganic surface of the

QD is inaccessible.55 As a result, the direct coordination of polyhistidine motif to the

inorganic shell of a QD is not possible for the preparation of QD-bioconjugates.

However, when the amphiphilic polymer exhibits carboxylic groups, the mutual

chelation of Ni2+ ions by surface carboxylic groups and a polyhistidine motif tag can

be used for self-assembly of biomolecules.56 In this bioconjugation strategy, the role

of surface carboxylic groups is analogous to NTA, which when associated with Ni2+

ions is known to coordinate strongly with the polyhistidine motif.32 Alternatively, in

the absence of surface carboxylic groups for polymer encapsulated QDs, the surface

of QDs can be modified with NTA, which when supplemented with Ni2+ ions, can be

used for polyhistidine mediated self-assembly of biomolecules.57 For a greater in-

depth discussion on bioconjugation using the polyhistidine motif, the reader is

referred to a review article by Blanco-Canosa and coworkers.32 In addition,

bioconjugation of QDs has been extensively addressed in recent reviews and

interested readers are referred to these review articles.53, 54

Control of adsorption of oligonucleotides on QDs

For the development of QD-FRET bioassays, QD surface coatings based on thioalkyl

acids (e.g. MAA, MPA and DHLA) are attractive given their compact ligand size, ease

of preparation of aqueous QDs and subsequent capacity for bioconjugation.21 As

thioalkyl acid ligands were among the first reported QD surface coatings to impart

aqueous solubility to QDs33, initial efforts by our group to develop QD-FRET nucleic

acid hybridization bioassays also investigated QDs coated with MAA and MPA

ligands.58, 59 It was found that the analytical performance of QD-FRET nucleic acid

hybridization bioassays was greatly affected by the adsorption of oligonucleotides

on MAA/MPA-capped QDs.58, 59 Given that non-specific adsorption is undesired in

bioassay development, Algar and Krull investigated the origin of adsorption of

oligonucleotides on MPA- and MAA-QDs using FRET as a transduction method.60, 61

The adsorption was investigated using Cy3-labeled oligonucleotide sequences and

green-emitting CdSe/ZnS QDs as donors. FRET is a useful tool to study adsorption of

oligonucleotides on QDs. This method only requires oligonucleotides to be labeled

with a suitable fluorescent acceptor dye that is paired with a QD donor, and the

strong dependency of FRET efficiency on the donor-acceptor separation distance

(Equation 1) allows differentiation between adsorbed and freely diffusing

oligonucleotides with high sensitivity and minimal perturbation of the system.61 The

caveats to using FRET to interrogate oligonucleotide adsorption include: that this

method is unable to differentiate between changes in FRET efficiency that originate

from changes in the stoichiometry of adsorbed oligonucleotides on the QD surface

and dynamic changes in the ‘tightness’ of adsorbed oligonucleotides on the QD

surface; and that the FRET efficiency response can potentially saturate prior to the

saturation of a QD surface with adsorbed oligonucleotides.61 Nonetheless, FRET is a

useful tool to study adsorption of oligonucleotides on the QD surface with the

expectation that conditions that suppress the number of oligonucleotides adsorbed

on the QD surface will also reduce the ‘tightness’ of oligonucleotide bound on the QD

surface.61 Thus, both of these effects will work in concert to decrease FRET

efficiency.

Figure 2. (a) Adsorption behavior of oligonucleotides on MPA-QDs. (i) Dependence

of the pH of buffer on the adsorption of Cy3-labeled mixed base sequence on MPA-

QDs. (ii) Changes in the Cy3/QD PL ratio (FRET ratio) for the adsorption of

increasing molar ratio of Cy3-dA20 sequence on MPA-QDs at pH 7.4. The inset shows

changes in the FRET ratio for the adsorption of Cy3-dC20, Cy3-dA20, Cy3-d(TGGG)5

and Cy3-dT20 on MPA-QDs at pH 7.4. (iii) Changes in the adsorption of Cy3-labeled

mixed base sequence on MPA-QDs as a function of increasing NaCl concentration at

pH 8.5 and pH 9.3. (b) (i) Chemical structure of a glutathione (GSH) ligand. Changes

in the FRET ratio for solution-phase hybridization of oligonucleotide probe modified

GSH-QDs with increasing number of (ii) 3' Cy3-labeled fully-complementary (FC)

proximal target and (iii) 5' Cy3-labeled FC distal target at pH 9.2. Note the difference

in the sensitivity response of (ii) and (iii). In (a), panels (i), (ii) and (iii) adapted with

permission from reference61. Copyright 2011 Elsevier. In (b), panels (ii) and (iii)

adapted with permission from reference19. Copyright 2014 American Chemical

Society.

As presented in Figure 2a (i), experiments involving titration of MPA-QDs (as

donors) with increasing numbers of acceptor dye-labeled oligonucleotides showed

that adsorption was strongest at acidic pH and decreased with increase in solution

pH.61 In the case of CdSe/ZnS QDs coated with MAA, adsorption was found to be

more than 10-fold greater at pH 4.8 than at pH 9.5.60 The pH dependent adsorption

experiments exhibited a profile analogous to an acid-base titration curve, showing

that the pKa of QD-bound MPA ligands was ca. 7.8.61 In contrast, the pKa of MPA in

bulk solution has been reported to be 4.3.62 This elevation of pKa of MPA at the QD

interface is consistent with a previously published study that has reported an

elevation of pKa of carboxylic group at the nanoparticle interface.63 Adsorption

experiments and competitive binding experiments showed that different

nucleobases exhibit varying degrees of tendency to adsorb on MPA-QDs. As shown

in Figure 2a (ii), the order for adsorption affinity of different nucleobases on MPA-

QDs was found to be dC > dA ≥ dG >> dT.61 The adsorption of oligonucleotides on

MAA- or MPA-QDs was found to be driven by hydrogen-bonding, where neutral

carboxylic groups of thioalkyl acid ligands interacted favorably with nucleobases.60,

61 Support for the hydrogen-bonding mechanism in mediating adsorption of

oligonucleotides on MAA- and MPA-QDs was provided by experiments that showed

that the addition of a hydrogen bond disrupter (i.e., formamide) suppressed

adsorption of oligonucleotides on the QD surface.60 In addition, the extent of

adsorption of double-stranded oligonucleotides was found to be significantly less

than single-stranded oligonucleotides.60 The extent of oligonucleotide adsorption on

MPA-QDs was also found to be dependent on the ionic strength of solution as shown

in Figure 2a (iii). A log-linear relationship between adsorption and ionic strength

was observed at pH 8.5, while at pH 9.3, where ionization of MPA ligands on QDs is

expected to be complete, adsorption was found to be negligible up to 100 mM NaCl

concentration.61

Adsorption also impacted the conformation of oligonucleotide probes conjugated to

the QD surface, and influenced hybridization kinetics and stability of duplex

formation at the QD interface.60 Hybridization of QD-probe conjugates with a fully-

complementary (FC) target that was labeled with an acceptor dye at either the distal

(5') or proximal (3') terminus offered similar FRET efficiencies, which were also

found to be independent of the linker length used for QD bioconjugation.60 This

suggested that at low density, oligonucleotides laid along the surface of QDs instead

of orienting upright from the QD surface. Increasing the number of conjugated

probes on the QD surface caused immobilized probes to orient upright from the QD

surface when subjected to hybridization with complementary targets.60 The rates of

hybridization of complementary oligonucleotides to the QD-probe conjugates were

found to scale proportionally to the rate of adsorption of non-complementary

oligonucleotide.60 In addition, adsorption impacted the thermodynamic stability of

DNA duplex at the QD interface.60 For a FC target, the hybrids at the QD interface

exhibited sharper melt curve transition and a decrease in melt temperature (Tm) by

2 °C as compared to the bulk solution hybridization.60 In contrast, hybrids

containing mismatches exhibited an increase in Tm and broadening of melt curve

transition as compared to bulk solution counterparts.60 Melt curves obtained under

the conditions in which adsorptive interactions became less favorable showed

transitions which closely resembled bulk solution hybridization.60 These effects

were attributed to the competition between probe-target interaction and

adsorption of oligonucleotides on the QD surface. The impact of adsorption

interactions on the stability of hybrid was found to be temperature dependent.60

Adsorptive interactions served to stabilize duplex formation below the Tm, while

they facilitated duplex denaturation above the Tm, resulting in sharper melt curve

transitions.60

The insights into the mechanism of oligonucleotide adsorption on thioalkyl acid

capped QDs were seminal in shaping further development and improving the

analytical performance of QD-FRET nucleic acid hybridization bioassays. In recent

studies, we have used glutathione capped QDs (GSH-QDs) for the preparation of

water soluble QDs and subsequently used these QDs for the assembly of QD-FRET

nucleic acid hybridization bioassays.17-20 As shown in Figure 2b (i), GSH is a

tripeptide exhibiting a thiol group, a primary amine group and two carboxylic

groups. The proximal thiol and primary amine groups coordinate to the ZnS shell of

CdSeS/ZnS (core/shell) QDs while the distal carboxylic groups under sufficiently

basic conditions provide colloidal stability in aqueous media. Given that the distal

carboxylate groups are responsible for aqueous solubility of GSH-QDs (pH and ionic

strength dependent), it was anticipated that the interaction of nucleobases with

neutral carboxylic groups of GSH via hydrogen bonding mechanism could

potentially contribute to the non-specific adsorption of oligonucleotides. The

adsorption of oligonucleotides on MAA- and MPA-QDs was suppressed under basic

conditions (pH > 9).61 By conducting the hybridization assays at pH 9.2 using GSH-

QDs that were modified with single-stranded oligonucleotides as probes, the non-

specific adsorption of oligonucleotides on GSH-QDs was sufficiently minimized that

no surface passivation of the QD surface was required.18, 20 It is likely that the partial

zwitterionic character of GSH also contributed to the suppression of oligonucleotide

adsorption on GSH-QDs.64 Hybridization bioassays conducted with a FC target that

was labeled with an acceptor dye at the proximal (3') or distal (5') terminus showed

significantly different assay sensitivities as can be seen in Figure 2b (ii) and (iii),

respectively, which was in contrast with MPA-QDs. The FC target labeled at the

proximal end showed ca. 100-fold higher assay sensitivity as compared to the FC

target that was labeled at the distal end.19 This provided further confirmation that

the oligonucleotide probes were not adsorbed on the surface of GSH-QDs. It is also

interesting to note that under the conditions where adsorption was favored on

MPA-QDs (pH 7.4), the ratio of the FRET-sensitized acceptor dye PL to QD donor PL

(FRET ratio) saturated at an acceptor to QD ratio of 4 to 1, as can be seen in Figure

2a (ii). In contrast, hybridization of GSH-QDs modified with oligonucleotide probes

with a dye-labeled FC target offered a saturation of FRET ratio response at an

acceptor to QD ratio of 40 to 1 for both the proximal (Figure 2b (ii)) and distal

(Figure 2b (iii)) labeled targets. This suggests that the suppression of

oligonucleotide adsorption on a QD surface also impacts the loading capacity of

oligonucleotides probes on the QD surface, where adsorption excludes some of the

surface area of a QD from oligonucleotide conjugation. The use of thiol ligands for

the development of homogenous QD-FRET nucleic acid hybridization assays has

also been reported by other groups65-67. In these studies, various thiol ligands were

appended to a PEG moiety to suppress non-specific adsorption of oligonucleotides

and to promote colloidal stability of QDs in aqueous medium.

QD-FRET assay using intrinsically labeled probes

Transduction of nucleic acid hybridization by a QD-FRET method relies on a change

in the positioning of an acceptor dye with respect to the donor QD surface upon

target hybridization. This results in a modulation of FRET efficiency response, which

serves as an analytical signal.68 From the standpoint of QD-FRET transduction of

nucleic acid hybridization, the proximity between the donor QD surface and the

acceptor dye has primarily been accomplished either by directly labeling a target

strand with the acceptor dye18, 20, 58, 59 or by introducing a sandwich hybridization

format that makes use of a labeled reporter strand11, 12, 17, 19. Although these

approaches are functional, they introduce additional processing steps, potentially

increasing the complexity of assays. Additionally, these approaches are strictly

limited to in vitro assay configurations (cf. ex vivo or in vivo QD-FRET transduction of

nucleic acid hybridization). Given that the detection of unlabeled targets is

desirable, our group has recently reported a homogenous assay format for QD-FRET

transduction of nucleic acid hybridization that made use of intrinsically labeled

oligonucleotide probes for the detection of unlabeled targets.69 The oligonucleotide

probe strands were labeled with two adjacent molecules of a derivative of thiazole

orange (TO) intercalating dye. The modified probes were then bioconjugated to the

surface of green-emitting QDs (QD525) using SAv-biotin for coupling.69 The QDs

served as donors for the excitation of TO fluorescent dyes by FRET. In the absence of

probe-target duplex formation at the QD interface, the two TO molecules formed an

H-aggregate dimer, resulting in quenching of the fluorescence emission of the dye

molecules due to excitonic interaction between the two dye molecules.69 Upon

hybridization, the H-aggregate dissociated as the dye molecules preferentially

intercalated with the double stranded DNA duplex, resulting in

restoration/enhancement of the fluorescence emission of the dye molecules. The

relative positioning of the dye molecules from the donor QD surface, the distance

between the two dye molecules and the attachment location (DNA phosphate

backbone or thymine nucleobases) greatly impacted the analytical performance of

the assay.69 The hybridization bioassays provided a limit of detection (LOD) of 10

nM (2 pmol) and a dynamic range spanning one order of magnitude, and this

performance was identical to targets of 34 and 90 nucleobase length.69 The

selectivity of the assay was shown by single nucleotide polymorphism (SNP)

discrimination. Albeit in its infancy at the current stage, with further development

and optimization, the use of intrinsically labeled probes in conjunction with the QD-

FRET transduction method may potentially allow direct probing of the dynamics of

intracellular gene expression levels.

Solution versus solid-phase RET bioassays

Advantages of solid-phase bioassays

Nanoparticle (NP)-based RET bioassays may be done with NPs dispersed in solution

(solution-phase RET) or immobilized on a solid substrate (solid-phase RET). Solid-

phase bioassays offer some opportunities beyond the solution-phase methods.

Nanoparticle immobilization in solid-phase bioassays eliminates the need to limit

the reaction conditions to only those that allow for colloidal stability of

nanoparticles. For instance, a high ionic strength condition may be applied to a

solid-phase assay to accelerate the reaction between similarly charged

nanoparticles and DNA, but the same high ionic strength condition may compromise

the stability of solution-phase nanoparticles. Bioconjugation onto a surface-

immobilized nanoparticle is greatly enhanced by applying a large excess of

biomolecules or biorecognition elements, and solid-phase immobilization allows for

washing to remove unbound molecules. Washing of the surface may also be

advantageous to remove interferences prior to the detection step. While washing of

NPs that are in solution-phase can be accomplished, the process requires tedious

and less efficient purification steps. Moreover, solid-phase bioassays offer

multiplexing potential by spatial arraying of probes on the solid-substrate, and

potentiate the use of near-field optical techniques such as surface plasmon

resonance spectroscopy and photonic crystal enhanced fluorescence.11

In addition to the general advantages of the solid phase bioassays, RET bioassays

may particularly benefit from the close proximity of immobilized NPs at an

interface. As discussed in section 1.3, the RET efficiency increases with the number

of acceptors assembled onto the nanoparticle donor. In a similar fashion, when

donors and acceptors are immobilized at a high density on a solid substrate, a single

acceptor may accept energy from multiple donors resulting in increased FRET

efficiency.20

Immobilization of nanoparticles on solid surfaces

A key step in the assembly of solid-phase RET bioassays is the immobilization of

nanoparticle donors on a solid substrate. Our group has investigated the

immobilization of nanoparticles on a variety of solid substrates including glass,

paper, and fused silica optical fibers.18 The goal was to develop surface chemistries

that are facile, that provide robust surface immobilization, and may potentially be

applied to a variety of substrates. For instance, thiol, imidazole and amine groups

are known to coordinate with the surface of CdSe/ZnS QDs,70 and carboxylate,

phosphate and amine groups are the most widely used anchoring groups for

immobilization of lanthanide-based UCNPs.71 Another strategy was to use selective

and high-affinity interactions of biomolecules for nanoparticle immobilization.

Early attempts to immobilize QDs made use of monothiol-functionalized fused silica

optical fibers as the substrate.18 The lability of binding between thiol and ZnS

surface of QDs contributed to instability when using monodentate thiol-

functionalized surface. Efforts then took inspiration from previous work that

addressed functionalization of QDs in bulk solution, and multidentate surface ligand

exchange (MSLE) was explored to enhance the stability of immobilization on

surfaces. The first MSLE embodiment included the functionalization of bidentate

DHLA groups (dithiol) on silica optical fibers.18 Next, the bidentate ligand was

replaced with a tetradentate thiol ligand to further enhance the robustness and

surface density of QD film on glass and silica substrates. 13 Despite the increased

coordination by multidentate thiol groups with the ZnS surfaces of QDs, the

immobilized QDs tended to dissociate from surfaces as the thiol groups oxidized

over time. The search for more stable chemistries then investigated multidentate

imidazole-functionalized surfaces for QD immobilization using MSLE. A variety of

substrates, including glass, polystyrene microtiter plates and paper substrates were

functionalized with multidentate imidazole groups and provided for stable QD

immobilization.18

Another strategy for QD immobilization was via high-affinity biomolecular

interactions such as DNA hybridization and Streptavidin (SAv)-biotin interactions.

As one example, QD immobilization was achieved via DNA hybridization between

two complementary oligonucleotides, one tethered to glass and the other

functionalized on QD surfaces.24 In another example, high-affinity binding between

SAv and biotin was used for immobilization of QDs on glass substrates for FRET

bioassays of DNA targets. 11, 24

Immobilization of UCNPs on solid substrates can also be achieved. This has usually

been reported in the literature as being driven via physical adsorption. We have

used streptavidin-coated UCNPs on paper substrates for Luminescence Resonance

Energy Transfer (LRET ) DNA hybridization bioassays.72 First, the oleate surface

ligands on UCPs were oxidized to render the nanoparticles ligand free and then

sodium citrate and streptavidin were sequentially coated on the surface. Although

physical adsorption provides a facile strategy for immobilization of UCNPs, weak

binding results in instability and desorption of immobilized nanoparticles.

Moreover, the low-density coverage of adsorbed nanoparticles obviates any RET

signal enhancement due to the interaction between neighboring RET pairs in

adjacent UCNPs. Therefore, we developed an strategy that allows for immobilization

of UCNPs onto modified paper substrates via covalent chemistry.73 Amine modified

UCNPs were prepared by ligand exchange using o-phosphorylethanolamine (PEA)

and subsequently immobilized on the aldehyde functionalized glass coverslips. The

closely-packed solid-phase UCNPs showed an improvement in assay sensitivity in

comparison with both solution-phase bioassays and also less densely packed solid-

phase bioassays. Improvement in sensitivity would result from greater availability

of surface area for selective reaction and potentially also from optical “cross-talk”

from nearest-neighbour interactions.73

QD immobilization in microfluidic channels

Microfluidics is a powerful tool in various applications in biology including

bioassays, bioconjugation, drug development and delivery, and tissue engineering.

Microfluidic chips present a promising platform for solid-phase RET bioassays with

significant advantages over the bioassays done in bulk solution. Such advantages

include a reduction of the quantity of sample and reagents required for bioassays

and improved speed, sensitivity, resolution and throughput of the bioassays.

However, immobilization of NPs by adsorption on the surface of microfluidic chips

does not necessarily provide sufficient stability. For instance, the applied electric

field in chips that operate using capillary electrophoresis and electro-osmotic flow

imposes significant force on the QD-oligonucleotide conjugates immobilized on

thiol-functionalized chip surface, and has been seen to cause desorption and

migration of nanoparticles.21 One successful strategy to create stable NP films on

glass microfluidic channels utilized DNA hybridization.24 The surfaces of the

microfluidic channels were covalently coated with single-stranded oligonucleotide

that served as a tether. Two different oligonucleotides were conjugated on QDs, one

of which was used as an anchor to hybridize to the complementary oligonucleotide

tether attached to the glass, and the other remained available to serve as a probe for

hybridization with target DNA that could be transported in the microfluidic channel.

This strategy allowed for analysis of target DNA using FRET, subsequent NP removal

by introduction of a denaturing conditions to dehybridize the tether-anchor system,

and re-use of the microfluidic chip by recoating with fresh QDs for subsequent

cycles of analysis of target oligonucleotides.

Another approach made use of SAv-biotin interaction for QD immobilization on the

surface of a glass-PDMS microfluidic chip for FRET bioassays of DNA targets.21

Electroosmotic flow (EOF) was used to deliver SAv-QDs through the microfluidic

channel to dynamically immobilize the NPs onto the biotin-functionalized glass

substrate. Subsequently, application of voltage was used to deliver biotin-

functionalized DNA probes to decorate the immobilized QDs. The FRET system was

then ready for determination of aliquots of Cy3-labeled oligonucleotide targets that

were transported by application of voltage. Two types of commercial SAv-QD

conjugates were used; SAv molecules were either conjugated directly or through a

PEG spacer to the QD surface. The FRET bioassays demonstrated that the QDs which

were directly conjugated with SAv provided a significantly higher FRET efficiency,

and this is consistent with provision of a closer proximity between QD surfaces and

the Cy3 molecule labels that served as acceptors.15 This strategy was extended to

explore a two-plex FRET hybridization assay using PL spatial profile of QDs of two

different color (QD525 and QD605) as donors and Cy3 and Alexa-647 as acceptors

(Figure 3).14

Figure 3. Schematic representation of multiplexed nucleic acid hybridization bioassays in a hybrid glass/PDMS based microfluidic channel using immobilized QDs as FRET donors. Adapted with permission from reference14. Copyright 2013 Elsevier.

Extending microfluidic methods for application to NP decoration

Microfluidics flow has been used for on-chip immobilization of NPs and subsequent

probe conjugation on NP surfaces.14, 15 The immobilization of NPs allows treatment

with a sequence of reaction and washing solutions, and laminar flow in microfluidic

environments offers excellent control of reaction conditions at surfaces that achieve

reproducibility with high speed. This suggests potential for use of microfluidic chips

as a manufacturing platform for NP decoration. Moreover, a particular advantage of

using NP decoration in microfluidic channels is the potential to achieve coatings on

defined areas of NP so that different molecules can be conjugated to one NP. When

NPs are strongly immobilized on a surface, one side of NPs is sterically blocked by

the surface. Thus, ligand immobilization may only occur on the solution-facing side

of the NPs. Subsequent removal of such NPs from the surface into solution exposes

the unmodified sides of the NPs that can be further derivatized. Asymmetrically

decorated (Janus) NPs may be produced using solid-phase decoration. Single-phase

microfluidics (SPM) based on continuous flow is limited as a platform for NP

decoration, as SPM is plagued by cross-contamination and slow mixing due to the

laminar nature of the flow. Another challenge for NP decoration using continuous

flow microfluidics is the scale-up, as the throughput only linearly increases with the

footprint of the device. Thus, reaching manufacturing scales requires substantial

increases in the numbers of channels on the chips.

Droplet-based microfluidics

One form of droplet-based microfluidics is based on injection technology that

produces sequential multiple discrete droplet volumes that are supported in a

flowing immiscible phase.,74. Various droplet manipulations, including merging,

splitting, sorting, trapping and pairing may be used to fulfill different functionalities

required for complex NP decoration processes. Compartmentalization of reactions

in individual droplets of small size enables more precise control over reaction

conditions, and the convective flow inside the droplets helps to speed reactions.

Moreover, the production in a manufacturing scale may be achieved by increasing

the rate of micro-reactor droplets without the need for increasing the device

footprint.

NP decoration requires several steps including addition of NPs and ligand solutions,

removal of excess reagent, washing and recovery of decorated NPs. We have

recently developed a solid-phase method for decoration of QDs and gold NPs with

DNA oligonucleotides.75 Negatively charged NPs are first electrostatically associated

onto the surfaces of positively charged magnetic beads (MBs), to create MB-NP

conjugates (Figure 4a). Negatively charged oligonucleotides are electrostatically

adsorbed onto the MB surfaces when added to a suspension of MB-NP conjugates.

This creates a high local oligonucleotide concentration at the surface of the MBs that

promotes the conjugation reaction. This oligonucleotide preconcentration effect has

been observed to result in conjugation of oligonucleotides onto NP at kinetic rates

increased by over 1000 fold in comparison to bulk solution reactions, and an

oligonucleotide surface density ~5 fold higher than the best achieved for QDs when

using solution-phase methods (Figure 4(b) and 1(c)). Decorated NPs were

subsequently released from MBs by changing pH and ionic strength. Every step of

the process, including MB-NP conjugation, DNA decoration and NP recovery was

complete in less than one minute. The process was entirely governed by

electrostatic forces, thus switching from one step to another only required a change

in ionic strength and/or pH. This method offers simplicity and speed, two important

criteria required for integration into the droplet microfluidic manufacturing

platform for decoration of NPs.

(a)

(c)

(b)

Figure 4. (a)Schematic representation of solid-phase QD decoration, and ligand

density quantification using a FRET assay. QDs were conjugated to the surfaces of

MBs to form MB-QDs, then DTPA-modified DNA was immobilized on QD surfaces.

Following the release of QD-probe conjugates from MBs, the probes were hybridized

with Cy3-labeled complementary targets. The density of surface-immobilized

oligonucleotides was monitored using gQD-Cy3 FRET assay. (b) and (c) show the

kinetics of DNA immobilization on QDs using solution-phase and solid-phase

methods , respectively. Adapted with permission from reference75. Copyright 2016

American Chemical Society.

QD-to-AuNP energy transfer

Significant efforts have been directed towards improving the energy transfer

efficiency in RET bioassays that use QDs. QDs offer numerous advantages as donors,

but one commonly cited drawback is the physical size of the nanocrystals as this

influences RET distance.76 Energy transfer originates from the center of the QD, and

the physical size of the QD and its coating limits the distance between an acceptor

and the donor. Given that distances may be relatively fixed, efficiency of energy

transfer might be improved by consideration of spectral overlap and by dipole

alignment. The use of metallic NPs as acceptors has been explored to improve RET

efficiency. Metallic NPs, with gold nanoparticles being the most popular, attribute

their unique optical properties to the phenomenon of localized surface plasmon

resonance (LSPR).77 These unique optical properties include aspects such as high

molar extinction coefficients and broad absorption spectra, making gold NPs

efficient acceptors in non-radiative energy transfer based bioassays. Furthermore,

the LSPR spectra of metal NPs can also be tuned by controlling the size, shape and

chemical composition.78

Numerous studies have demonstrated the utility of gold NPs as acceptors in energy

transfer bioassays that involve QDs as donors, and these have recently been

reviewed.79 Such bioassays typically involve immobilization of biorecognition

elements (e.g. DNA, antibodies, peptides) on the surface of both the donor and

acceptor NPs. Selective interactions between the biorecognition elements and the

target molecules are designed to bring the two nanoparticles together, facilitating

energy transfer. Many of the early efforts to develop such bioassays involved the use

of very small gold nanoparticles (diameter – 1.4 nm), which are commercially

available under the brand name of Nanogold.80 The popularity of this material was

facilitated by its commercial availability as a monofunctional nanoparticle.

Monofunctionality ensured that each nanoparticle was conjugated to a single

biomolecule/ligand. This permitted its precise arrangement around a single QD,

without the complication of forming cross-linked aggregates. Such structural control

becomes particularly important when assembling responsive/functional multi-

nanoparticle constructs that respond to the presence of a target molecule. In large

aggregates, the nanoparticles/recognition elements near the center of the complex

may experience a different environment than those that exist at the periphery. This

can result in non-uniform response to target molecules. The use of small AuNPs

enabled controlled conjugation onto QDs, and also permitted greater energy

transfer efficiencies than traditional fluorophores or quenchers.81

The use of larger AuNPs (≥ 3nm diameter) should increase energy transfer

efficiencies due to the presence of stronger plasmon resonance bands. However, the

increased surface area of larger AuNPs inhibits facile monofunctionalization, and

provides for a greater number of permutations in which the nanoparticles may

assemble. In fact, most bioassays that have used AuNPs larger than 3 nm in diameter

were not designed to form responsive multi-nanoparticle complexes, but rather

relied on the aggregation of nanoparticles in the presence of a target molecule to

bring together the QDs and AuNPs in order to quench the QD photoluminescence.82,

83 For these bioassays, the functionality or responsiveness of the multi-nanoparticle

complex was not of concern, and thus the requirements for design were not

stringent. Only a few studies have used larger AuNPs as part of a target responsive

multi-nanoparticle complex84. Another challenge encountered with use large gold

nanoparticles is an inner filter effect. The strong extinction coefficients of these

AuNPs tends to block the excitation and emitted radiation, decreasing the signal

intensities that are usually observed.84 A decrease in analytical performance is

typically observed when large AuNPs are used.

Our group recently demonstrated that difficulties encountered when implementing

AuNPs with sizes greater than 3 nm, namely the inner filter effect and the large

surface area, may be overcome by the use of monofunctionalized QDs.85 By

monofunctionalizing the QDs, the large surface area of the AuNPs no longer presents

a challenge as each QD may only bind to one AuNP. Furthermore, this configuration

enables the assembly of multiple QDs around a single AuNP, decreasing the

concentrations of AuNPs that need to be used, which consequently reduces the

impact of the inner filter effect. In the study, two different configurations were

tested as shown in Figure 5. Configuration 1 involved monovalent QD-DNA

conjugates placed around a single AuNP that was functionalized with multiple

copies of a complementary DNA sequence. Configuration 2 involved placing

monovalently functionalized AuNP-DNA conjugates around a QD functionalized with

a complementary DNA sequence. A competitive DNA displacement reaction was

used to manipulate the separation between the QDs and AuNPs, enabling the

investigation of energy transfer interactions for the two configurations.

Furthermore, the influence of the inner filter effect was also evaluated for the two

different configurations by monitoring fluorescence intensities of identical QD

concentrations in the two different configurations. For configuration 1, three

different sizes of AuNPs (6 nm, 13 nm and 30 nm; diameters) were investigated,

while for Configuration 2, a 6 nm AuNP was used as the acceptor. The performances

of the bioassays were measured on the basis of increase in fluorescence intensity as

the QDs were separated from the AuNPs using the DNA strand displacement

reaction. Based on the results of the study, placing fifteen QDs around a 13 nm AuNP

(Configuration 1) provided optimal performance. To obtain a similar response using

Configuration 2, three 6 nm AuNPs had to be placed around a single QD. While

energy transfer efficiencies were similar, the absolute fluorescence intensities of

Configuration 1 was five times greater than that of Configuration 2, allowing for

better overall analytical performance as gauged by emission intensity. The

difference in absolute fluorescence intensities is attributed to the reduced inner-

filter effect when smaller concentrations of AuNPs are used in Configuration 1 as

compared to Configuration 2. This becomes especially important with the growing

trend of using low-cost detectors, such as cell phone cameras, to collect optical

signals for sample analysis.86

Figure 5. Evaluating assay configurations to optimize the design of QD-AuNP based

energy transfer bioassays. The schematic diagram serves to depict the multi-

nanoparticle structures formed in solution. The graph demonstrates the increase in

fluorescence intensity as a function of increasing amount of target DNA, which is

added to dissociate the QDs from the AuNPs. Adapted with permission from

reference86. Copyright 2015 American Chemical Society.

Over the course of the study, two main deficiencies with handling the nanomaterials

were identified. Firstly, to optimally design multi-nanoparticle complexes, the

concentration of the nanomaterials must be accurately identified so that the

complexes generated are reproducible. Nanoparticles do not have a defined

molecular weight and this is a consequence of the variability of composition,

dispersity in size and the distributions of arrangements of stabilizing ligands that

cover the nanoparticles. This results in an uncertainty in their molar concentrations.

Significant effort is being directed towards developing methods for the

quantification of nanoparticles, and these were recently reviewed.87 Most current

methods are only applicable to large nanoparticles (>10 nm), with small

nanoparticles such as QDs being challenging to quantify. A novel technique

represented in Figure 6 was developed by our group to determine the molar

concentration of small nanoparticles such as QDs.88

Figure 6. Schematic diagram summarizing the method used to determine

nanoparticle concentration. Nanoparticles are first functionalized with a suitable

ligand at low equivalences (<2). The distribution of nanoparticle-ligand complexes

are then quantified using an appropriate technique to construct the graph

presented. The mathematical correlation of the ligand distribution to the ligand

concentration provides the number of nanoparticles present in solution. Adapted

with permission from reference88. Copyright 2015 American Chemical Society.

The technique relied on quantitative analysis of the nanoparticle-ligand distribution

that exists when nanoparticles are functionalized with small equivalents of ligands.89

In this method, nanoparticles were modified with small equivalents (<2) of PEG

based ligands, with the different nanoparticle-ligand complexes being separated

using agarose gel electrophoresis. Densitometric analysis of the gel images allowed

for the quantification of the conjugates of different valences. Fitting this

nanoparticle-ligand distribution to a statistical function (Poisson model) enabled

the determination of the number of ligands per nanoparticle. This information,

along with accurate knowledge of the ligand quantities and reaction efficiencies,

permitted the calculation of nanoparticle concentration. The technique was

validated using gold nanoparticles and binary QDs (CdSe/ZnS; core/shell), and was

found to be in excellent agreement with standard methods of analysis. The

technique was then applied to alloyed QDs to determine their extinction coefficients,

which was previously unknown. It was these alloyed QDs that were subsequently

used in the experiments to optimize assay configurations for QD-AuNP based energy

transfer bioassays.

The second challenge that was identified was the generation of monovalent

biomolecule-nanoparticle conjugates. This is a consequence of nanoparticles having

a large surface area, providing them with multiple reactive sites with equivalent

reactivity.90 This often leads to a distribution in the valency of nanoparticle-

biomolecule conjugates even if functionalized with low equivalences of

biomolecules. Monovalent conjugates are currently obtained using one of two main

approaches. One involves the concept of electrostatic repulsion 91 to minimize the

number of biomolecules immobilized per nanoparticle. The second and more

common strategy is to purify the monovalent conjugates from a mixture of

conjugates.92, 93 Purification methods enjoy the advantage of being applicable to

nanoparticles with different surface chemistries and conjugation protocols. Our

group developed a facile technique for the isolation of QDs that are

monofunctionalized with a single DNA strand as illustrated in Figure 7.94

Figure 7. Schematic diagram summarizing the isolation of monovalent QD-DNA

conjugates using magnetic beads. The DNA modified QDs were captured onto

diethylaminoethanol (DEAE) functionalized magnetic beads using electrostatic

interactions. Washing with solutions of specific ionic strength (controlled using

sodium chloride) allowed for the selective elution of monovalent conjugates.

Fractions containing monovalent conjugates were identified using agarose gel

electrophoresis. Adapted with permission from reference94. Copyright 2014

American Chemical Society.

Magnetic beads with positively charged functional groups were used to capture QDs

functionalized with DNA at different valences. Selective elution of the QD-DNA

monoconjugates was then achieved by tuning the ionic strength of a wash solution.

Isolation of monoconjugates, prepared with nucleic acids as short as 19 bases in

length, was achieved with high efficiencies (≥ 70%). The wide applicability of this

method was demonstrated by its use to purify monovalent conjugates of

commercially available water-soluble QDs and also monovalent AuNP-DNA

conjugates. The popularity of magnetic beads in separation science has facilitated

the development of numerous automated platforms for magnetic bead handling

with high-throughput. These advantages may now be applied towards the

purification of monovalent nanoparticle conjugates.

UCNPs as donors in resonance energy transfer applications

Upconversion mechanisms

The study of upconversion nanoparticles (UCNPs) has grown rapidly over the last

two decades as there is interest to access unique optical and chemical

characteristics. Unlike most fluorescent materials that produce emission of photons

with lower energy than used for excitation, UCNPs are a class of material that emits

photons with higher energy than those used to excite the NPs. The practical

advantage for assay design of this optical feature is primarily in the ability to use

low energy photons in the near-infrared region of the spectrum to ameliorate

scatter and autofluorescence caused by the excitation radiation, while achieving

emission at UV and optical wavelengths. UCNPs offer another important advantage

in contrast to QDs, and this is the emission of multiple wavelengths in narrow bands,

rather than only one wavelength that is emitted by QDs. The process of

upconversion can be achieved by at least five different mechanisms.95 For a detailed

discussion of these mechanisms, the reader is directed elsewhere.95 The most

efficient of these mechanisms, electron transfer upconversion (ETU), is of greatest

interest for development of RET donors with sufficient quantum yield to develop

sensitive detection systems. In the ETU mechanism (Figure 8), the energies from

multiple photons are absorbed by a lanthanide emitter (activator) ion through

energy transfer from adjacent lanthanide sensitizer ions that are excited by photons

into metastable states. As sequential excitation of potentially emissive states in the

emitters become populated, relaxation of the promoted electrons results in the

emission of higher energy photons by combination of the energies of excitation

photons.96 The emission from lanthanides is not formally a fluorescence process,

and is instead referred to as a luminescence process, leading to the terminology of

Luminescence Resonance Energy Transfer (LRET).

Figure 8. An illustration of the energy transfer upconversion mechanism. Adapted

with permission from reference96. Copyright 2009 Royal Society of Chemical.

UCNP design and synthesis

Both the sensitizer and activator ions are doped inside an inorganic crystalline host

lattice. The choice of host lattice plays a direct role in the efficiency of the energy

transfer process. The crystal structure determines the spacing between the

sensitizer and activator ions, and thus the energy transfer efficiency.97 The degree of

symmetry around the cationic centers influence whether the ions will undergo f-f

Laporte forbidden transitions or favor radiationless decay pathways. Other factors

such as low lattice phonon energy and lattice matching of the sensitizer and

activator ions also need to be considered in the design of highly luminescent and

homogenous upconversion nanoparticles.96

With the lanthanide ions able to emit upconverted radiation, attention can be

directed to the RET process. As with any RET experiment, the choice of the donor-

acceptor pair is crucial in the overall energy transfer efficiency of the system. When

choosing a suitable RET acceptor, all of the conditions of wavelength overlap,

distance of the donor and acceptor, and dipolar alignment described in Section 1

still apply. UCNPs can be synthesized to produce a wide range of emission bands

across the UV to NIR spectral region, providing some opportunity to tune an

emission wavelength of the donor to be in strong resonance with an acceptor. The

emission wavelengths are constant intrinsic properties of the lanthanides in the

local crystal environment. However, by varying the identity as well as the

percentage composition of the dopants and their host lattices, different emission

profiles can be obtained. For example, in the NaYF4:Yb3+/Pr3+ system, it was

observed when changing the Pr3+ (activator) concentration from 0.1-1%, the ratio of

the blue to green emission changed almost 2 fold.98 This is because when more Pr3+

ions were present in the host lattice, the average distance between the ions was

reduced, allowing for more cross-relaxation events to take place. This in turn

decreased the population of the 3P0 state that was required for the blue emission.

Tuning of the intensities of emission bands of UCNPs by varying the concentrations

of activators to sensitizers has been demonstrated as a facile method for

synthesizing UCNPs with different dominant colors of emission.99 In another report,

a NaYF4:Yb3+/Ho3+ system was co-doped with 0-15% Ce3+ to tune the emission

intensity from green to red by a factor of over 10 fold.100 This was achieved due to

the relatively small energy gap which exists in Ce3+ that directs Ho3+ activators to

transfer energy from green emitting pathways to red emitting pathways. Another

approach to tuning the emission is by controlling the size of the UCNP. This is more

difficult to achieve since it requires nanoparticles in the range of 5-10 nm, and the

reproducible synthesis of UCNPs of diameters less than 10 nm seems challenging.101

However, these methods have their own drawbacks as the former requires specific

ligands that can be difficult to exchange when constructing the RET system, and the

latter requires more complex manipulation of instrumentation.

A host lattice composed of NaYF4 is one of the best for upconversion

nanomaterials.102 For such UCNPs, two phases are commonly observed, α and β,

with the β phase providing higher quantum yield due to symmetry where there are

two independent sites within the crystal lattice for the sensitizer and activator,

leading up to a four-fold increase in the energy transfer from the sensitizer to the

activator.4 There are a number of approaches for synthesizing UCNPs of different

morphologies and compositions, and these have been reviewed extensively.96, 103 A

commonly used method to synthesize β-NaYF4 UCNPs is hydrothermal synthesis

where the starting materials, salts of sodium, lanthanides and fluorides are

dissolved into a solvent, such as water, and placed in an autoclave. Typically, NaOH

or NaF, lanthanide salts of nitrates, oxides and chlorides, and NH4F are used as

precursors for the reactions. In this method, a Teflon coated autoclave is often used

to provide an inert environment where the desired reaction can occur. The solution

is heated above the critical point of the solvent for an extended period of time.104

The advantages of this method include the relatively inexpensive starting materials

and lack of intermediary steps involved in the reaction. Another common synthesis

method is thermal decomposition.105 High boiling point solvents such as oleic acid,

oleylamine and octadecene are used to dissolve lanthanide precursors before a

solution containing Na+ and F- is added using a low boiling point solvent such as

methanol. The methanol is then boiled off before the temperature of the solution is

raised above 300 °C for 20 to 60 min. Although this method requires inert

atmosphere and more expensive starting materials compared to the hydrothermal

method, it allows for the production of high quality UCNPs with narrow dispersity of

size and well defined morphology.105

Applications of UCNP as donors in RET

UCNPs can be used as RET donors with organic dyes serving as acceptors. The

excitation wavelengths of many fluorphores are in the UV-visible region of the

electromagnetic spectrum, while UCNPs are typically excited with near-infrared

radiation. This is advantageous as there is no concern that NIR radiation will

directly excite the acceptor molecules. Also, since many organic dyes are susceptible

to photobleaching when exposed to intense UV-visible radiation, the use of NIR

excitation allows for acceptor emission to be integrated over significant periods of

time. A further important attribute for assay development depends on the intensity

of radiation in various emission bands. The multiple emission bands from a single

type of UCNP offers potential for multiple targets to be detected simultaneously. For

example, UCNPs have been used as donors in RET to detect two different DNA

sequences by using the green channel of NaYF4:Yb/Er as the donor for Cy3 and the

red channel as the donor for Cy5.5.106 In this work, two different DNA probe

sequences were attached to the UCNP surface, and complementary sequences were

labelled with either Cy3 or Cy5.5. Both targets could be detected selectively even

when at sub-picomole quantities.

In a separate example, Li and coworkers designed a Ca2+ probe using the quenching

of UCNP emission with fluorescein being the RET acceptor.107 Fluo-4, a commercial

fluorescein based Ca2+ receptor was directly conjugated to the UCNP surface and the

emission of the UCNP at 490 nm was quenched up to 80% after conjugation. This

was a result of multiple fluorescein molecules behaving as RET acceptors of the

UCNP donors. When the Fluo-4 coated UCNPs were introduced into an environment

containing Ca2+, the emission at 490 nm was recovered in a concentration

dependent manner. The researchers then used this nanoprobe to detect Ca2+ in the

cytosol of live cells as well as in cancer tissues. This example illustrates how RET can

be used as a ratiometric method to quantitatively determine target concentrations,

and that recovery of donor signal as a result of the loss of RET can provide useful

analytical information. One pratical consideration for all RET methods that use NP

donors is that the selective chemistry contributes to the colloidal and

thermodynamic stability of the NPs in solution, and the properties of the surface

coating can change if selective binding proceeds.

UCNP-to-QD energy transfer

QDs offer photostability, high extinction coefficients, and narrow emission bands

suitable for multiplexing. However, their broad absorption profiles can present a

challenge. Due to the typical Stokes shift emission of fluorescent material, the

wavelengths that are used to excite the donor will concurrently excite the QDs. The

use of QDs as acceptors has therefore been limited to chemiluminescence resonance

energy transfer (CRET) and bioluminescence resonance energy transfer (BRET)

where no excitation source is used.108, 109 QDs have also been used as acceptors with

lanthanide complexes as donors in time gated measurements where it is possible to

separate emission from excitation by means of time.110 However, the use of UCNPs

that are excited in the NIR and that exhibit anti-Stokes emission as donors, avoids

the direct excitation of QDs. The shorter wavelength emissions from UCNPs,

typically blue or green, overlap well with the absorption bands of QDs that extend

from their first exciton peak to UV wavelengths. Numerous studies have made use of

an UCNP-QD LRET pair, and include tunable multicolour structures, optical

bioassays and photovoltaic devices. Energy transfer efficiencies from UCNP to QDs

have also been studied.

Li et al. encapsulated QDs in the silica shell of NaYF4 UCNPs, where QD fluorescence

was generated by LRET from the UCNP cores. The multiplexing capacity was

increased by doping the shell with different colors of QDs. QD605-doped

silica/NaYF4:Yb,Tm nanospheres were used as luminescent probes in cell imaging

using a confocal microscope equipped with 980 nm NIR laser. Luminescence from

cells was observed with high S/N and minimal autofluorescence. The signal from the

nanoparticles was increased with increasing output power from the NIR laser, while

the noise remained low.111 Yan et al. reported NaYF4 UCNPs that were decorated

with CdS QDs. The combination of upconverting and semiconducting properties

resulted in the appearance of sub-band-gap photoconductivity. The creation of an

electron-hole pair in QDs using sub-band-gap energies by first exciting the UCNP

overcomes fundamental limitations of single junction photovoltaic devices. This

demonstrates the potential use of UCNPs in photovolatic devices to harvest photons

with sub-band-gap energies.112

Bednarkiewicz et al. were the first to characterize LRET between UCNPs and QDs in

detail. The NPs were placed in close proximity by drying a mixture on a microscope

slide. The average lifetime of UCNP emission decreased from 153 to 130 µs,

indicating LRET between UCNPs and QDs. The Förster radius was calculated to be

15 Å with an energy transfer efficiency of 14.8%. The short distance was attributed

to the low quantum yield of UCNPs.113 A study by Mattsson et al. reported a Förster

radius of around 4.6 – 6.0 nm and a LRET efficiency of 7%. The large Förster

distance was attributed to the strong spectral overlap between UCNP emission and

QD absorbance, and the large molar extinction coefficient of QDs. Streptavidin-to-

biotin binding brought the NPs in close proximity for LRET. Since a large number of

Er3+ ions were in the core of the UCNPs and beyond reach of RET, there was minimal

decrease in UCNP luminescence at 550 nm in LRET. To determine the change in

lifetime of Er3+ ions close to the surface of the UCNP, the lifetime of these ions in the

absence of LRET was compared to the lifetime of QD fluorescence in LRET. A change

from 79 to 74 µs was seen. A lifetime component of 374 µs remained unchanged

and was attributed to the Er3+ ions in the core of the UCNP.114

Doughan et al. reported solid phase bioassays using NaYF4 UCNPs and QDs for the

detection of thrombin on microscope slides and nucleic acids on paper. This early

work demonstrated the covalent immobilization of UCNPs on glass. The UCNPs were

decorated with a thrombin specific aptamer to capture thrombin that was then

labelled with a second QD conjugated aptamer. This served to bring UCNPs and QDs

in close proximity for LRET.73 The immobilization chemistry was transferred onto

paper and the functional UCNPs were used for the detection of nucleic acids. Limits

of detection of 13 fmol were achieved with high selectivity and a contrast ratio of

fully complementary target to one base pair mismatched target of 60.5 in 10%

formamide. The assay was tested in 90% goat serum samples and achieved limits of

detection of 24 fmol within 3 minutes.115 Recently, Doughan et al. reported a

triplexed assay on a paper substrate for the simultaneous detection of three nucleic

acid sequences using green, orange and red QDs as acceptors. The narrow and well

defined emission peaks for both NPs allowed the collection of luminescence from all

NPs using only optical band pass filters in an epi-fluorescence microscope. Limits of

detection of 26 fmol were achieved.

UCNP-to-AuNP energy transfer

AuNPs can also be combined with UCNPs to engineer new materials and bioassays.

Recently, Huang et al. fabricated a hybrid core/shell nanocomposite consisting of a

gold nanorod (GNR) core and an upcoverting NaYF4: Yb, Er shell. The GNR plasmon

was tuned to be in resonance with the red Er3+ emission allowing a single excitation

source at 980 nm to be used. By creating hybrid nanostructures, high efficiency and

localization are achieved. The green emission peak from the Er3+ is in poor

resonance with the plasmon of the GNR and was found to be temperature

dependent. This was used as an optical thermometer to measure local temperature

since the green emission is a two-photon absorption process where distribution in

the occupancy of the electronic states follows the Boltzmann distribution.116

Gold nanomaterials have also been used as quenchers of UCNP luminescence in

biosensing. Lin et al. reported an aptasensor for platelet-derived growth factor

(PDGF-BB), which is over expressed in some human tumours. Gold NPs of 7 nm size

provided strong absorption at 520 nm that was in strong overlap with the green

emission from NaYF4:Yb,Er nanoparticles. A sandwich assay structure with two

PDGF specific aptamers was used to bring UCNPs and AuNPs in close proximity for

LRET. The use of an NIR excitation source minimized autofluorescence and allowed

for good selectivity in blood serum of lymphoma patients. A decrease in the

luminescence from UCNP was observed suggesting the presence of PDGF-BB.117

Since the transmission of UV-visible light is attenuated in biological samples, Chen et

al. utilized NIR to NIR upconversion for the detection of mercuric ions. Gold

nanorods (GNRs) were used as quencher with a strong absorption band that

overlapped with 804 nm emission from the NaYF4: Yb, Tm UCNPs. The presence of

Hg2+ combined UCNPs and GNRs modified with Hg2+ specific aptamers. The assay

reached limits of detection of 2 nM in water and 10.4 nM in human serum. 118 The

same group reported a turn-on assay for the detection of -fetoprotein, a cancerα

marker. GNRs were attracted to anti-AFB decorated NaYF4: Yb, Tm/NaGdF4 UCNPs

electrostatically, which quenched UCNP emission in the NIR region of the spectrum.

In the presence of AFP, GNRs were released restoring UCNP emission. A limit of

detection of 0.16 ng mL-1 was reported for bioassays using clean samples.

Measurements determined using diluted serum samples were comparable to those

determined from standard clinical methods.119

Summary and Future Directions

The use of inorganic nanoparticles in RET bioassays provides a number of

advantages compared to systems that use organic dyes. Key among these are

attributes such as resistance to photobleaching, sharp emission bands, substantial

Stokes and anti-Stokes shifts and the potential for multiplexing across wavelength

channels. The decoration of nanoparticle surfaces with various types of ligands have

been shown to render the nanoparticles water soluble, and can provide for selective

binding events to take place. RET bioassays have been shown to be functional in

solution, and in the solid-phase, with glass, plastic and paper as typical solid-phase

platforms. Distance dependency of RET can be a limiting factor for larger NPs and

when implementing thicker surface coatings. Opportunity to improve RET efficiency

may be realized by using clusters of NPs and cascades of RET relay centers for co-

operative energy transfer and collection. Although work has been done to determine

the number of DNA molecules on gold NPs and QDs, the loading density of different

types of ligands can be challenging to control and is not always straight forward to

determine. As different ligand chemistries and manufacturing methods are

explored, the ability to better control the packing density as well as the conjugation

of mixtures of ligands on NP surfaces will further mature. These advances would in

turn lead to improvement of the variety of targets for these NP-based bioassays

including more intracellular bioassays, as well as allow for design of the effective

concentration range for quantitative determination of target molecules. Increasing

the structural stability of the modified NPs over prolonged storage time and

integration with sample preparation steps on convenient substrates such as paper

platforms would allow the technology to penetrate into clinical, agricultural and

environmental settings.

Acknowledgements

We are grateful to the Natural Sciences and Engineering Council of Canada for

research support (Grants STPGP 479222-15 and RGPIN-2014-04121), and for a

graduate fellowship awarded to SD.

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Author Biographies:

Yi Han received his B. Sc. degree in Chemistry from the University of British

Columbia, Vancouver, BC, Canada in 2010. He is a graduate student under the

supervision of Prof. Ulrich J. Krull and is pursuing research in the development of

UCNPs for DNA detection on solid-phase platforms. Prior to joining the Chemical

Sensors Group, he has worked on a diverse range of projects in biochemistry and

materials chemistry.

Omair Noor received his Hon. B.Sc. degree in Biotechnology in 2008, and M.Sc. and

Ph.D. degrees in bioanalytical chemistry, in 2010 and 2015 respectively, under the

supervision of Prof. Ulrich J. Krull from the University of Toronto. He is currently a

research and development scientist at the ACGT Corporation (Toronto, Canada)

working in the area of nucleic acid diagnostics. Omair has received numerous

awards that include the TalentEdge industrial postdoctoral fellowship from the

Ontario Centres of Excellence, Ontario Graduate Scholarship from the Ontario

Ministry of Training, Colleges and Universities and Undergraduate Student Research

Award from the Natural Sciences and Engineering Research Council of Canada.

Abootaleb Sedighi has been a postdoctoral fellow at University of Toronto, Canada

since 2015. His current research under the supervision of Prof. Ulrich Krull focuses

on development of nanomaterials for diagnostic and theranostic applications. He

received his Ph.D. degree in Chemistry from Simon Fraser University, Canada. In his

Ph.D. research under supervision of Prof. Paul C.H. Li, he used nanoparticles to

modify DNA hybridization reactions. He received his M.Sc. degree from Shahid

Beheshti University in 2008.

Uvaraj Uddayasankar completed his BSc in biological chemistry (2009) at the

University of Toronto Mississauga, followed by an MSc (2010) and a PhD (2015) in

bioanalytical chemistry at the University of Toronto. The focus of research during

his doctoral studies was on investigating the interactions between quantum dots

and gold nanoparticles for energy transfer based assays. In 2016, he moved to

Houston Methodist Research Institute for a post-doctoral position where he was

involved in the validation of point of care devices for clinical diagnostics. Currently,

he is training to become a clinical biochemist at the University of Manitoba. His

research interests focus on facilitating the implementation of nanomaterial based

analytical assays for clinical diagnostics.

Samer Doughan received his Hon. B. Sc. degree in Chemistry and Mathematical

Sciences in 2012 from the University of Toronto Mississauga, Mississauga, ON,

Canada. He is currently working towards his Ph. D. degree under the supervision of

Prof. Ulrich J. Krull at the University of Toronto. His research interests focus on the

use of UCNPs in LRET based assays for the multiplexed detection of proteins on

solid supports. Samer currently holds a Post Graduate Scholarship (PGS-M) from the

Natural Sciences and Engineering Research Council of Canada (NSERC). He has

previously received Ontario Graduate Scholarship (OGS) from the Ontario Ministry

of Training, Colleges and Universities (MTCU) and Undergraduate Student Research

Award (USRA) from NSERC.

Ulrich Krull is a professor of analytical chemistry at the University of Toronto

Mississauga (UTM). He holds the endowed AstraZeneca Chair in Biotechnology. His

research work explores biosensors, bioassays, immobilized biomolecules,

microfluidics and nanomaterials, to advance technology with applications in

clinical/medical, forensic, agricultural and environmental areas. Krull is an editor of

Analytica Chimica Acta, a leading journal for analytical chemistry.

Authors: Back L to R; Krull, Sedighi, Doughan, Han; and Front L to R Noor, Uddayasankar

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