Fluorescent nanosensors: rapid tool for detection of food contaminants

FLUORESCENT NANOSENSORS: RAPID TOOL FOR DETECTION OF FOOD CONTAMINANTS

20

Bhaswati Bhattacharya*, Siddhartha Singha*, Santanu Basu** *National Institute of Food Technology Entrepreneurship and Management, Kundli, Haryana, India; **Panjab University, Dr. S. S. Bhatnagar University Institute of Chemical Engineering & Technology, Chandigarh, India

1 Introduction Fluorescence is emission of characteristic UV-visible electromagnetic radiation by molecules excited by electromagnetic radiation of a particular wavelength. This phenomenon has been the basis of various analytical techniques across the field. For ­example, fluorescent probes like tailor-made dyes are quite popular for in vivo marking of cells for disease detection, efficacy study of drug molecules, structural evaluation of biological materials, and so forth. However, the traditional fluorescent molecules are not always useful in complex biological system due to their susceptibility toward molecular environment and their tendency of photo-bleaching. As an alternative, nanoparticles with capability of fluorescence have been found useful in bioassays. Currently the food industry is feeling pressing needs of employing sensitive, selective, and robust nanosensors owing ­ to conscious customers, demanding regulatory bodies, issues like bioterrorism. Therefore, food processing and food science is ­driving the study of a potential fluorescent nanoparticles, a.k.a fluorescent nanosensors. Miniaturized biosensors based on fluorescence principles are slowly gaining commercial importance for rapid detection of food contaminants, such as, pesticide residues, foodborne pathogens, and their toxins. In past two decades the spectroscopic method or imaging techniques have seen a very rapid pace of development. Evolution of laser-based imaging systems such as stimulated emission Nanobiosensors. http://dx.doi.org/10.1016/B978-0-12-804301-1.00020-5 Copyright © 2017 Elsevier Inc. All rights reserved.

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842  Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants

depletion, photo-activated localization microscopy, and stochastic optical reconstruction microscopy have enabled the measurement of various parameters of fluorescence such as intensity, decay time, and polarization, effects of resonance energy transfer, quenching, or photo-induced electron transfer. In sync with the development of the measurement devices, the capacity of synthesizing novel nanomaterials with customized fluorescence properties has expanded to a great extent. A large number of nanoparticles with silica or modified silica, hydrophobic and hydrophilic organic polymers, semiconducting organic polymers, quantum dots, carbon (quantum) dots, nanoclusters and nanotubes, nanodiamonds, metal and metal oxides particles, and so forth, have been synthesized and characterized by different workers. They differ in the method of synthesis, surface plus fluorescence properties, and application. Hence, an elaborate discussion of all of these nanosensors is beyond the scope of a single chapter. This chapter on fluorescent nanosensors thematically discusses the fluorescent-based quantum dots for food contaminant detection and is broadly divided into two thematic areas. The first part of the chapter introduces the concept of fluorescence for smooth sailing into the main concept of fluorescent nanosensors. The second part elaborates synthesis, characterization, and application of variety of quantum dots with fluorescence.

2  What Is Fluorescence? 2.1 Phenomena The emission of light from the electronically excited states of molecules is termed as luminescence. Depending upon the nature of the excited states from where the emission is taking place, luminescence can be either fluorescence and/or phosphorescence. In case of excited singlet states, the electron is oppositely spin-paired with the electron in the ground-state orbital and henceforth, transition takes place from excited singlet state to ground state, which is spin allowed resulting in photon emission. In most cases, fluorescence is observed due to aromatic compounds. A few common examples of fluorescent compounds generally termed as fluorophores are depicted in Fig. 20.1. One of the most explored fluorophores is quinine, which is available in tonic water. If the tonic water is exposed to sunlight then a faint blue glow is observed on the surface. This light is more prominent if the observation is taken at right angle with respect to the direction of sunlight and in a solvent with low dielectric constant. Sir John Frederick William Herschel was the first person to report in 1845



Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants  843

Figure 20.1.  Typical fluorophores used for sensing: fluorescein and rhodamine.

(Herschel, 1845) that quinine solution gives fluorescence. There are several other fluorophores that are encountered in daily life. Antifreeze substances show a green or red/orange glow, which is due to the presence of trace amount of fluorescein or rhodamine (Fig. 20.1). Compounds, such as, anthracene and perylene also shows structured fluorescence and these compounds can be used to monitor oil pollution. Fluorescence is known for high sensitivity detection. In 1877, fluorescence was exploited to establish that the Danube and Rhine rivers are interconnected by underground streams (­Berlman,  1971) Fluorescein was added in Danube and after several hours green fluorescence of fluorescein appeared in a small river that flows to the Rhine. The history of fluorescence is provided in detail by B ­ erlman (1971). The plot of fluorescence intensity against wavelength (­nanometers) or wavenumber (cm–1) would give a fluorescence emission spectrum. Fig. 20.2 depicts two types of fluorescence emission spectra. The chemical structure of the fluorophore as well as the solvent in which the fluorophore is dissolved dictates the nature of the emission spectra. In the case of perylene,

Figure 20.2.  Emission spectra of perylene and quinine.

844  Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants

the emission spectra is structured due to the distinct vibrational energy levels of the ground and excited states, whereas other compounds like quinine show spectra devoid of any kind of vibrational structure.

2.2 Principle and Characteristics 2.2.1  Jablonski Diagram The different molecular processes that can occur between the different electronic states of a molecule can be represented by the Jablonski diagram (Jablonski, 1935). The diagram can be of d ­ ifferent forms to illustrate the different photophysical processes that occur. Professor Alexander Jablonski has been regarded as the father of fluorescence spectroscopy after this. Fig. 20.3 shows a representative Jablonski diagram. The ground, first, and second singlet electronic states are depicted as S0, S1, and S2 respectively. Again, the fluorophores can exist in the different vibrational energy ­levels of a single electronic energy level. The transitions between the different energy states are shown as vertical lines to depict that absorption

Figure 20.3.  Jablonski diagram depicting different electronic states and photophysical phenomena.



Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants  845

of light is instantaneous in nature. The transitions are in the order of femtoseconds, which is a measurement too small for significant displacement of nuclei as per Franck–Condon principle. Both of the processes—that is, absorption and emission—­ occur from the lowest vibrational energy state. Thermal energy is not sufficient to populate the excited vibrational states at room temperature, so light energy is required to induce fluorescence from S1. There are several processes that can occur after absorption of light by the molecule. In most cases, a fluorophore is excited to higher vibrational levels of either S1 or S2. The molecules then immediately relax to the lowest vibrational level of S1 in a time period of 10–12 s or even less and this process is termed as internal conversion. Internal conversion takes place prior to emission as the fluorescence lifetime is in the order of 10–8 s. Thus, emission occurs from the lowest vibrational energy level of S1, which is a thermally equilibrated excited state. There are possibilities of spin inversion where the molecules from S1 state can go to the first triplet state T1. The emission from T1 is termed as phosphorescence and appears at longer wavelengths with respect to fluorescence. The process of movement of the molecules from singlet state to triplet state is called as intersystem crossing. Rate constants of triplet emission are much smaller than fluorescence as transition from T1 to S0 is spin forbidden. Phosphorescence quantum yields can be increased by the presence of heavy atoms, such as, bromine and iodine.

2.2.2  Stokes Shift Fluorescence is observed at lower energies or longer wavelengths as compared to absorption and is shown in Jablonski diagram (Stokes, 1852). In case of all fluorescent molecules in solution there is considerable energy loss between excitation and emission. The molecules once excited rapidly decay to the lowest vibrational level of S1 that results in Stokes shift. In addition to the above, the molecules further loose excess vibrational energy as they decay to lowest vibrational levels of S0 from higher vibrational levels of singlet ground state. Further Stokes shift can be observed due to the effect of solvents, reactions in the excited states, complex formation, energy transfer, and so forth. Michael Kasha (Kasha, 1950) stated that the fluorescence spectrum remains same irrespective of the excitation wavelength. After the fluorophores are excited to the higher electronic and vibrational energy levels, they immediately return to the lowest vibrational level of S1, dissipating the excess energy. Hence, due to this fast relaxation process, the fluorescence emission spectra are normally independent of the excitation wavelength.

846  Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants

2.2.3  Fluorescence Quenching The decrease in the intensity of fluorescence is called quenching. In the excited state when a fluorophore is deactivated by some other molecule (commonly known as a quencher) due to contact or collision, then it is referred to as collisional quenching. The fluorophore returns to the ground state on interaction with quencher molecule and the process is diffusion-controlled. There is no chemical alternation of the molecules during such quenching phenomena. The Stern–Volmer Eq. 20.1 describes collisional quenching: F0

F

= 1 + K [Q ] = 1 + kqτ 0 [Q ]

(20.1)

Here, K = Stern–Volmer quenching constant, kq = the bimolecular quenching constant, τ0 = initial lifetime, [Q] = the concentration of the quencher. The Stern–Volmer quenching constant K highlights the level of interaction of the fluorophore with the quencher. When a fluorophore is embedded deep inside a macromolecule then it is highly inaccessible to the water-soluble quenchers and then the K value is lower, whereas if the fluorophore is free or on the surface of macromolecules, then K is quite high. The quenching mechanism depends on the fluorophore and the quencher. When acrylamide quenches the emission of indole group in tryptophan then the quenching is due to the electron transfer from indole to acrylamide in the excited state. Halogen and heavy atoms cause quenching through spin-orbit coupling and intersystem. Apart from collisional quenching there exist other processes through which fluorescence quenching can take place. Sometimes, fluorophore molecules form complexes with the quenchers in the ground state that are nonfluorescent. This type of quenching is called static quenching as it occurs in the ground state and is not affected by diffusion or molecular c­ ollisions.

2.2.4  Resonance Energy Transfer (RET) This process occurs when the emission spectrum of a fluorophore called as donor overlaps with the absorption spectrum of another molecule known as acceptor (Förster, 1948) illustrated in Fig. 20.4. The aforementioned absorption process is dependent on the concentration of the acceptor. In the RET process, there is no involvement of intermediate photons. The donor and acceptor molecules are coupled by a dipole–dipole interaction and the extent of energy transfer is given by the spectral overlap and



Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants  847

Figure 20.4.  Illustration of FRET spectrally and through Jablonski diagram.

the distance between the donor and acceptor. The rate of energy transfer is expressed as Eq. 20.2:



kT (r ) =

1  R0  τ D  r 

6

(20.2)

Here r is the distance between the donor (D) and the acceptor (A) while τD is the lifetime of the donor without energy transfer. Spectral overlap is described in terms of the Förster distance (R0). The efficiency of energy transfer for a single donor–acceptor pair at a certain fixed distance is given as Eq. 20.3: E=

R06 R06 + r 6

(20.3)

The aforementioned expression suggests that the extent of energy transfer depends on distance (r). The Förster distances are generally comparable in size to the biological macromolecules and thereby this RET has been used as a spectroscopic ruler for measurements of distances between the different active sites on proteins (Fig. 20.5). Depending upon the nature and linkage of donors and acceptors, the theory of RET can vary. Donors and acceptors can be covalently linked, can be free moving in solutions, or may be

Figure 20.5.  Illustration of FRET as a “spectroscopic ruler.”

848  Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants

confined in macromolecules. The extent of energy transfer may increase depending on the lifetime of the donor molecule.

2.2.5  Fluorescence Lifetime and Quantum Yield The two most significant features of a fluorophore molecule is the quantum yield and fluorescence lifetime. The lifetime determines the time available for the fluorophore to interact with or diffuse in its environment. The rate of fluorescence emission of the fluorophore (Γ) and the rate of its nonradiative decay to S0 (knr) gives a measure of the quantum yield of the fluorophore. Both the rate constants Γ and knr depopulate the excited state. The fluorescence quantum yield is given by the ratio of number of photons that are emitted to the number of photons absorbed. A fraction of the fluorophores relaxes through emission and the quantum yield is expressed as Eq. 20.4:



Q=

Γ Γ + knr

(20.4)

The quantum yield can be close to unity if the nonradiative decay rate is substantially smaller than the radiative decay rate, that is, knr < Γ. There can be different nonradiative processes, such as, internal conversion, intersystem crossing, and all these nonradiative constants, which are grouped as knr. Stokes losses decrease the energy yield of fluorescence less than unity. The fluorescence lifetime can be defined as the average time that the molecule resides in the excited state prior to return to the ground state. It can be represented as Eq. 20.5:



τ=

1 Γ + knr

(20.5)

In absence of any nonradiative processes, the fluorescence lifetime measured is termed intrinsic lifetime and is given as Eq. 20.6:

τn =

1 Γ

(20.6)

Theoretically, the natural lifetime τ n of a fluorophore can be calculated from its absorption spectra, extinction coefficient and the emission spectra. In reality, natural lifetime can be determined from the measured lifetime (τ) and quantum yield as given in Eq. 20.7:

τn =

τ Q

(20.7)



Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants  849

In case if the rate constants (Γ or knr) are affected by certain factors, then both fluorescence lifetime and quantum yield get modified. For example, a large rate of internal conversion or slow rate of emission can make a molecule nonfluorescent. Biochemical fluorophores behave in an unpredictable manner unlike their unsubstituted aromatic molecules, if a fraction of fluorophores is located closer to quencher groups, as happens in the case of tryptophan residues in proteins, then there will be inevitable discrepancies. This leads to poor agreement between calculated τ from Eq. 20.7 and that calculated from its absorption and emission spectra.

2.2.6  Steady State and Time-Resolved Fluorescence In steady-state fluorescence measurements, the sample is i­lluminated with a continuous beam of light, and the intensity or emission spectrum is recorded. Most of the measurements are steady state due to the nanosecond time scale of fluorescence. A molecule quickly reaches to the steady state once it is exposed to light. The second kind of measurement is time-resolved for ­measuring intensity decays and/or anisotropy decays. In the case of time-resolved studies, the sample is irradiated with a light pulse, where the pulse width is shorter than the decay time of the molecule (Fig. 20.6) and this time-resolved intensity is then recorded with a high-speed detection system, where intensity is measured in the nanosecond timescale. The intensity decay of a fluorophore that have a single decay time (τ) is given as Eq. 20.8:

I (t ) = I 0 e −t τ

(20.8)

Figure 20.6.  Illustration of fluorescence life-time principle. The short-pulsed excitation light [dark gray (red in the web version)] and the longer time duration fluorescence emission light [light gray (green in the web version)] is shown as a function of time.

850  Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants

Where I 0 is the intensity at t = 0 immediately following the excitation pulse. Again, steady-state intensity is the average taken over the time-resolved intensity decays of the sample and can be expressed as Eq. 20.9: ∞



I ss = ∫ I 0 e −t τ dt = I 0τ

(20.9)

0

The value of I 0 depends on the concentration of fluorophore and other instrumental parameters. Eq. (20.9) reflects that the steady-state intensity is proportional to the lifetime of a fl ­ uorophore.

2.3  Fluorescence Imaging and Sensing Fluorophores act as an indicator (or probe) to determine intrinsically nonfluorescent species and other parameters, such as, pH, oxygen, or metal ions. The photoluminescent properties (fluorescence or phosphorescence intensity, anisotropy, lifetime, or emission wavelength) respond to the chemical composition of the environment. Luminescence probes can be configured on different basic principles and can be categorized as per sensing mechanism: (1) quenchable probes, (2) fluorogenic probes, (3) dual wavelength probes, (4) FRET-based probes, and (5) photoinduced electron transfer (PET) systems. The functionality of the fluorophore determines the kind of imaging method. Fluorescence quenching is one approach in use for chemical sensing and imaging. In due course of a chemical reaction, luminescence amplification is observed for fluorogenic probes. The “turn on” luminescent probes have proven to be advantageous over quenchable probes. There are also dual-wavelength probes, which are categorized under sensitive dyes that can be applied to intrinsically referenced assays. There are PET systems where the probes exhibit shifts in their absorption and/or emission maxima on protonation or formation of a complex with metal ions. Typically, chemical sensors are known to display two important features: (1) these sensors should contain a chemical or molecular recognition system (receptor), which is connected in series with a physicochemical transducer (Thevenot et al., 1999); and (2) they should be miniaturized devices that are able to disclose real-time and online information of the presence of certain specific compounds or ions even in complex biological samples (Cammann et al., 1996). Luminescent probes can often be encapsulated in nanoparticles to enhance the delivery module and thus also could be detected by a remote monitoring methodology. Thus, optical ­



Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants  851

chemical sensors should consist of the recognition unit (in a few cases, the chromophore itself can act both as a receptor and the transducer), a light source, and a photodetector. Fluorescent sensors are made of polymer materials where the indicator is incorporated and permeable to the target analyte.

2.4 Instrumental Methods for Sensory Imaging The instrumentation for sensory imaging consists of: (1) an indicator or a sensor layer, (2) an electronic system based on optics to record the photoluminescence of the indicator or the sensor layer, and (3) a control unit connected to the electronic system with image processing software. Such kind of imaging systems has wide application range from macro- to microscopic level. These systems have three components, a light source, such as a halogen lamp, light-emitting diodes, or laser diodes and optical filters in order to separate the short wavelength excitation light from the long wavelength luminescence light. In some compact instruments a beam splitter (dichroic mirror) is used, which reflects the short wavelength excitation light in sample direction while transmitting the long wavelength luminescence to the detector. Laser scanning and imaging, these are the two basic approaches that are in use for detection of analyte. The scanning principle is exploited in the confocal microscopes and in microarray readers. In general, scanners are equipped with various lasers for different excitation wavelength, a movable x/y stage and a photomultiplier tube (PMT) as a detector. On the other hand, a typical imaging system would consist of a CCD camera as the detector along with a LED array or white light source in combination with a set of appropriate filters (Schäferling, 2012). The use of fluorescent sensors in imaging is sometimes associated with certain disadvantages, such as, photobleaching of the dyes, background fluorescence of the sample, inhomogeneous illumination and instability of the light source, scattering of light, nonuniform distribution of the fluorescent probes in the sample analyte and also a varying thickness of the sample or sensor layer. Hence, the utmost requirement is that the optical sensor system should be thoroughly calibrated and referenced. Internally referenced techniques like fluorescence lifetime imaging (FLIM) are frequently employed, as it records the fluorescence lifetime, which is probably the most attractive intrinsically referenced parameter. The fluorescence lifetime as per Eq. 20.8 is not affected by the concentration of the fluorophores, static quenching effects, or the brightness of the light source used. In contrast, dynamic quenching, resonance energy transfer, and temperature have a strong

852  Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants

influence on the fluorescence decay. Thereby, the fluorescence lifetime is a preferred parameter in fluorescence sensing and imaging. In recent times, nanosensors based on fluorescence came into the field with wide applicability, which extended even to the detection of contaminants in the food material. In the forthcoming sections, sensors based on nanomaterials will be discussed with special focus on semiconductor-based nanomaterials, which are termed as quantum dots.

3  Nanotechnology-Enabled Sensors: Quantum Dots To meet the increasing demands of industry, new approaches based on nanotechnology have completely changed the area of sensor technology. This technology is enabling the development of small, inexpensive, and highly efficient sensors with wide applications. It is envisaged that by enhancing the interactions that occur at the nanoscale, nanosensors offer significant advantages over conventional sensors. This may be in terms of greater sensitivity and selectivity, lower production costs, and reduced power consumption, as well as improved stability. The unique properties of nanoscale materials make them ideal for sensing and such materials can be integrated into existing sensing technologies or can be made into new devices. This technology not only enhances the properties of existing materials but also enables the fabrication of novel materials and whose properties can be tailored specifically for sensing applications. There are possibilities for developing nano-bioorganic elements that are suitable for intracellular measurements. For ­sensing applications, nanotechnology allows development of nanostructures and the possibility of forming features, and which cannot be imagined with conventional microtechnologies. Such sensors are more favorable for sensing than the classically fabricated systems. Sensitivity may increase in such cases due to ­tailored conduction properties, the limits of detection may be lowered, infinitely small quantities of samples can be analyzed, direct analyte detection becomes possible sometimes without using labels and specificity may get improved. Improved sensitivity is a major attraction for developing nanotechnology coupled sensors. At the extreme nanoscale limit, there exists the potential to detect single molecule or atom. The small size, light weight, and high surface-to-volume ratio of nanostructures are the best candidates for the improvement in the detection of chemical and biological species with high sensitivity. Additionally in nanostructures the entire structure can



Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants  853

be affected by the analyte and not only the surface as c­ onventional sensors. As far as selectivity is concerned, nanoscale sensors and materials may not implicitly result in greater selectivity; however, nanostructuring materials and applying surface modifications and functionalization may greatly assist. The speed with which species can be detected is affected by the sensor’s dimensions. Nanostructures minimize the time taken for a measure and to ­diffuse into and out of that volume. Hence, nanoscale modifications present the opportunity for also improving the sensor’s dynamic performance. Nanosized spherical ­luminescent materials are applied as biomolecular labels in FRET assays. The incorporation of such photoluminescent materials into rigid polymeric matrices laid the platform for new biomolecular labels with higher brightness, good photostability, and substantial chemical stability. Moreover, with these kinds of modifications, the fluorophores get protective shielding from oxygen and other metal ions present in the environment that can cause quenching of the fluorophore. Such probes are also less affected by changes in pH and other chemical interferences.

3.1 Optical Nanosensors These are modern analytical tools that bring together the advantages of customary sensor technology with the flexibility of using dissolved indicators. They are defined as devices with dimensions smaller than 1000 nm that have the capability to incessantly monitor chemical or biological parameters by optically converting the available information into signal useful for analytical purposes. The dimensions of optical NSs vary from a few nanometers, for instance, for macromolecules with sensitivity up to 1 µm. Miniaturization of many fiber-optic sensors to submicron size has been achieved. Moreover, optical fibers impregnated with nanomaterials like carbon nanotubes have been successfully applied. Wide-ranging interest in surface-plasmon resonance (SPR) and localized surface plasmon laser (LSPR) spectroscopy has been reawakened by the availability of new fabrication methods for plasmonic materials. Optical nanosensors can be based on macromolecules, polymer materials, and sol–gels, multifunctional core–shell systems, multifunctional magnetic beads, metal beads, and quantum dots (Borisov and Klimant, 2008).

3.2 Nanosensor Materials Nanoparticles (NPs) can be classified on the basis of their composition as shown in Table 20.1 (Khanna, 2012).

854  Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants

Table 20.1  Classification of Nanomaterials According to Composition Sl No

Class

Examples

1

Metal-based

Metallic NPs, such as, Ag, Au, metal oxides such as zinc, or titanium oxides.

2

Carbon-based

Carbon nanotubes, fullerenes

3

Semiconductor-based

Quantum dots

4

Polymer-based

Dendrimers

5

Composite

Nanoclays, biomolecules combined with nanoparticles to form nanosized biocomposite

3.2.1  Core/Shell Structured Nanoparticles These are nanostructures that have the core made of a m ­ aterial coated with another material (Fig. 20.7). They have a size range of 20–200 nm. The necessity to shift to core/shell NPs arises from the quest for improvement in the properties of base NPs. ­Taking into consideration of the size of the NPs, the shell material is chosen is such that the agglomeration of particles is prevented that ­results in improving the monodispersity of the particles. The core/shell structure also enhances the thermal and chemical ­stability of the NPs, improves their solubility, makes them less ­cytotoxic (­poisonous to living cells), and allows conjugation

Figure 20.7.  Single multilayer core–shell nanostructures. From Liwei Su, Yu Ling, Zhen Zhou, 2011. Nanoscale 3, 3967–3983; Kochuveedu, S.T., Son, T., Lee, Y., Lee, M., Kim, D., Kim, D.H., 2014. Sci. Rep. 4, 4735.



Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants  855

of other molecules to these particles. In some cases, the shell also prevents the oxidation of the core material. When a core NP is coated with a polymeric layer or an inorganic layer like silica (SiO2), synergistically emerged functions can be envisioned because the polymeric or the inorganic layer would endow the hybrid structure with an additional function/property on top of the function/property of the core. The most widely used core/shell nanocomposites are gold or silver core with silica shell. The gold/silica NPs are used in optical sensing, and the thickness of the silica coat alters the property of gold nanoparticles. In semiconductor NPs, the core is made of semiconductor material, semiconductor alloy, or metal oxide with shell made of semiconductor material, metal oxide, or an inorganic material like silica. These structures can be binary (with a core and shell) or ternary (with a core and two shells). Quantum dots (QDs) are one of the most common binary structures. These are alloys of group III or group V metals or group IV and group VI metals: CdSe/Cds, CdSe/ZnS, ZnSe/ZnS, CdTe/CdS, and so forth; the shell thickness determines the emission range of these particles.

3.3  Quantum Dots These represent promising alternatives to organic fluorophores (chemical groups responsible for fluorescence). These are colloidal semiconductor nanocrystals, 1–10 nm in diameter, containing typically 103–104 atoms, because quantum confinement takes place in all three spatial dimensions (Wang and Herrron, 1991; Bruchez et al., 1998; Murphy and Coffer, 2002). The reduction of the size and dimensionality of metals results in a drastic change in the electronic properties as the spatial length scale of the electronic motion is reduced with decreasing size. When one dimension of a metallic material becomes comparable to the de Broglie wavelength (the wavelength of a particle given by λ = h/p, where h is Planck’s constant and p is momentum), the quantum confinements appear. In a semiconductor particle of this size, when an electron is promoted to the conduction band from the valence band, a hole is generated in the valence band with its own charge (+1) and effective mass. The electron and hole are bound to each other via coulombic forces of attraction, and this quasiparticle is known as exciton. The exciton is considered a hydrogen-like system and the spatial separation of the electron–hole pair can be calculated using the Bohr approximation of hydrogen atom.



r=

εh 2 π mr e 2

(20.10)

856  Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants

Where r is the radius of the sphere, ε is the dielectric constant of the semiconductor, mr is the reduced mass of the electron–hole pair, h is Planck’s constant, and e is the charge on electron. For many semiconductors, the masses of the electron and hole have been determined by ion cyclotron resonance and are generally in the range 0.1–3 me (me is the mass of the electron). For typical semiconductor dielectric constants, the calculation suggests that the electron–hole pair spatial separation is ∼1–10 for most semiconductors (Gaponenko, 1998). The electronic structure of these quantum dots becomes intermediate between localized and delocalized bands, and there is a change of electronic and optical properties of the material due to such quantum confinement The quantum dot is a good example of “particle-in-a-box” and the energies of the particle in the box depend on the size of the box. In the quantum dot, the band-gap energy becomes size dependent and is evident in simple absorption spectra of quantum dots in solutions (Fig. 20.8). The band-gap energy of the semiconductor spectra from such spectra is generally taken as the absorption energy onset. As the particle size decreases the absorption onset, blue-shifts indicate an increase in band-gap energy. QDs possess good brightness, high photostability (unchanged by the influence of light), broad absorption (radiation enters in a wide range of wavelengths) that allows for their simultaneous excitation, and relatively narrow emission. Application of QDs in

Figure 20.8.  (a) The energy band structure of Cdse crystals as size increases from small quantum dot and then to bulk semiconductor. (b) Absorption spectra of different-sized CdSe quantum dots. (Part a) From Kilmov, V.I., 2003. Los Alamos Sci. 28, 214–220. (Part b) From Tian J., Cao, G., 2013. Nano Rev. 4, 22578.



Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants  857

optical sensors can, however, be compromised by a high degree of nonspecific binding and by the fact that many substances act as quenchers (decreasing the fluorescence intensity of a given substance). Minimizing the interferences QDs can be used to successfully design sensors.

3.3.1  Classification of Quantum Dots Composition: On the basis of composition, quantum dots are classified as primary (first stage), binary (composed of two parts or two pieces), and ternary (composed of three items). Primary QDs are those made of single elements such as silicon QDs. Binary QDs are composed of two elements, such as, CdS, CdSe, and so forth. Ternary QDs contain three elements, such as, CdSeTe, ZnCdSe, and so forth. The use of semiconductor nanocrystals is potentially problematic because the high surface area of the nanocrystal might lead to reduced luminescence efficiency (ratio of energy of emitted photon to that of exciting photon, assuming that every exciting photon yields an emitted photon), and photochemical degradation. Controlling or altering the band-gap of a nanomaterial by changing the composition has led to the development of core–shell nanocrystals with high quantum yields at room temperature and with much improved photochemical stability. By enclosing a core nanocrystal of one material with a shell of another having a larger band-gap, the excitation can be confined to the core, eliminating nonradiative relaxation pathways and preventing photochemical degradation. Provision of chemical protection to the core, particularly against oxidation, is an added advantage of the shell. Surfaces of the quantum dots are sometimes capped with molecules or ligands to serve a few purposes: (1) partial passivation of dangling bonds on the surfaces of QDs; (2) preventing agglomeration of QDs through steric hindrance (the prevention or retardation of inter- or intramolecular interactions by the blockage of access to a reactive site by nearby groups; it arises from the crowding resulting from spatial structure of a molecule); (3) imparting solubility to the QDs. The ligands generally have two functional groups, one group binding with the QD and the other group interacting with the environment. A QD coated with surfactant molecules presents hydrocarbon chains to the environment while the polar head groups of the surfactant associate with the QD surface. Consequently, a surfactant coated QD is water insoluble but is easily dispersed in nonpolar organic solvents like toluene or hexane.

3.3.2  Optical Properties: Quantum Dots as Fluorescent Labels If the surface of the quantum dot is sufficiently passivated, it can have quantum yields of light emission that approaches to

858  Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants

those of organic dyes (∼0.6). Labels or tags constitute the foundations of luminescence and fluorescence imaging and sensing. Typical QDs are core–shell or core only structures functionalized with different coatings. Their properties depend to a considerable extent on particle synthesis and surface modification. Addition of a passivation shell often has the effect of producing a small red shift in absorption and emission relative to the core QD due to tunneling of charge carriers into the shell. QDs have the striking property of an absorption that gradually increases toward shorter wavelengths and a narrow emission band of mostly symmetric shape. The spectral position of absorption and emission are tunable by particle size (quantum size effect). The general principle for QD biofunctionalization is to make QDs water dispersible and then to be bound with biomolecules. Currently, only a few standard protocols are available for labeling biomolecules with QDs and the choice of suitable coupling chemistry depends on surface functionalization. QDs are promising in microarrays (multiplex lab-on-a-chip device), immunoassays and fluorescence in situ hybridization (the use of labeled nucleic acid sequence probes for the visualization of specific DNA or RNA sequences). Owing to the optical properties of organic dyes, their suitability for multicolor signaling at single-wavelength excitation is limited. QDs are the perfect contenders for spectral multiplexing at a single excitation wavelength because of their inimitable flexibility in excitation and their very narrow and symmetric emission bands, which simplify color discrimination. Organic dyes and the majority of fluorophores have poor photostability; for QDs, the microenvironment effect on spectroscopic features is mainly governed by the accessibility of the core surface. This again depends on the ligand and the strength of its binding to the QD surface and the shell quality. If no ligand desorption occurs, properly shelled QDs are minimally sensitive to microenvironment polarity. QD emission is scarcely responsive to viscosity, contrary to that of many organic dyes and QDs are also less susceptible to aggregation-induced fluorescence quenching.

3.4  Quantum Dots as FRET-Based Probes FRET involves the nonradiative transfer of fluorescence energy from an excited donor particle to an acceptor via dipole–dipole interaction through space (Fig. 20.9). FRET is commonly suited to measuring changes in distance, instead of absolute distances, appropriate for measuring protein conformational changes, monitoring protein interactions, and assaying of enzyme activity. QDFRET sensors are based on the interactions between QDs serving



Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants  859

Figure 20.9.  QD-based FRET probes displaying the displacement of a quenching dye by an analyte thus resulting in recovery of QD emission.* From Shamirian A., Ghai A., Snee P.T., 2015. Sensors 15(6), 13028–13051.

as donors and molecular fluorophores attached to the QD surface, acting as fluorescent acceptors. QDs have been exploited as FRET donors with organic dyes as acceptors, with the QD emission sizetuned to match the absorption band of the acceptor dye. Through FRET mechanism, QDs respond indirectly to environmental changes without any chemical interaction that could affect their photophysical properties and degrade their brightness. Several QD-based FRET strategies have been developed for detecting nucleic acid, proteins, and other small molecules. Owing to the free choice of the QD excitation wavelength, cross-talk is circumvented in such FRET pairs. However, the distance dependence of FRET means that both the size of the QD itself and that of the surface coating change the FRET efficiency and that typically renders QD-FRET less efficient as compared to FRET with organic dyes. Owing to the substantial size of QDs, this limitation can be partly overcome by increasing the number of adjoining small organic acceptor dyes.

3.4.1  Synthesis of Quantum Dots The most common and general method is to make colloidal solutions or to grow on solid substrates. In the colloidal approach, precursors of the material are reacted in the presence of a stabilizing agent that will restrict the growth of the particle and keep it within quantum confinement limits. For example, aqueous Cd(II)

860  Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants

salts can be mixed with anionic or Lewis basic polymers such as sodium polyphosphate or polyamines and the subsequent addition of a sulfide source produces CdS nanoparticles that are in the 1–10 nm size range. QDs may be manufactured with diameters ranging between broad limits starting from a few nanometers to as large as few micrometers. Size tuning is possible by controlling relative concentrations and rates of addition. QD synthesis can be tailored to meet specific requirements, with core, shell, and coating characteristics all affecting photochemical properties. Size distributions are controllable within 2% using accurate growth techniques, involving high annealing temperatures.

3.4.2  CdSe/ZnS Core/Shell QDs CdSe-ZnS core/shell QDs are prepared using synthetic techniques involving growth and annealing of organometallic precursors at high temperature. This method (Wang et al., 2009) is as follows: 1. A mixture of tri-n-octylphosphine oxide and hexadecyl amine is heated. Cadmium acetate is added to the solution. 2. The stock solution of trioctylphosphine-selenid is prepared by dissolving 0.2 g selenium in 5 g of tri-n-octylphosphine (TOP). 3. This stock solution is injected quickly into the reaction solution along with vigorous stirring of the same. Then nucleation of CdSe nanocrystals takes place. 4. Zinc acetate and bis(trimethylsilyl) sulfide are added for the inorganic epitaxial growth of the shell on the surface of the core. This epitaxial layer growth is done for about 2 h. CdSe/ZnS QDs with diameter of ∼3 nm, thus synthesized, dissolved in chloroform, and preserved in a sealed condition. Riegler et al. ( 2008) described another method: Cadmium stearate and TOP-Se are reacted at temperatures above 200°C by fast injection of TOP-Se into a mixture of tri-n-octylphosphine oxide and cadmium stearate. The CdSe cores are passivated and annealed by growing a shell of two additional monolayers of ZnS on their surface.

3.4.3  Quantum Dots and Organic Fluorophores The potential of detection to a great extent is determined by the physicochemical properties of the chromophore in use (Mason, 1999; Waggoner, 2006; Lakowicz, 2006) and that includes its chemical nature and size, its biocompatibility, and the effective interplay between the chromophore and the biological unit. Fluorophore properties affect the detection limit and the dynamic range of the method, and the aptness for multiplexing, that is,



Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants  861

parallel detection of different target analytes. Vital properties of a good label should be as follows (Resch-Genger et al., 2008): 1. It should be conveniently excitable, without excitation of the biological matrix at that instant, and the label should easily be detectable. 2. It should have a high molar absorption coefficient at the excitation wavelength with a high fluorescence quantum yield. 3. It should be soluble in relevant buffer solutions, cell culture media or body fluids. 4. It must be stable under conditions of usage. 5. The label should have necessary functional groups for site-specific labelling. 6. Its availability in reproducible quality must be ensured. 7. Whenever required, its deliverability into cells should be possible without any toxic effects. Organic fluorophore-based diagnostics is a versatile and widely practiced technology but there are certain difficulties faced with conventional fluorophores in multiplexed environment. As mentioned earlier, in comparison to organic dyes, QDs have an absorption that increases toward shorter wavelengths (below the first excitonic absorption band) along with a narrow symmetric emission band. The spectral positions of absorption and emission are tunable by particle size and width of the emission peak is determined by QD size distribution. The broad absorption enables free selection of the excitation wavelength and clear separation of excitation and emission. The size-dependent molar extinction coefficients at the first absorption band of QDs (100,000–1,000,000 M–1cm–1) (Yu et al., 2003) are generally large as compared to organic fluorophores (25,000–250,000 M–1cm–1). Fluorescence quantum yields of adequately surface-passivated QDs are normally high (0.65–0.85 for CdSe (Wang et al., 2003; Talapin et al., 2004), ≤0.6 for CdS (Spanhel et al., 1987), and 0.1–0.4 for InP (Xu et al., 2006). There are also examples of emitters, such as, CdTe and CdHgTe that have high fluorescence quantum yield in the visible-NIR wavelength (Shavel et al., 2006) and PbS and PbSe for the NIR wavelength region (Hinds et al., 2007; Du et al., 2002). On the other hand organic dyes have high fluorescence quantum yield in the visible range but they fall short in the NIR wavelength range with poor photo stability. Moreover in case of QDs, long lifetimes as compared to organic dyes enable temporal discrimination of the signal from autofluorescence and scattered excitation light by time-gated measurements, thus enhancing sensitivity. One of the important aspect of labels that it should not precipitate or aggregate under experimental conditions. In case of organic dyes solubility can be tuned via substituents without

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hampering the optical properties and other relevant features of the dye. On the other hand, QD dispersibility is controlled by the chemical nature of the surface coating. Although, for example, CdTe is inherently dispersible in water but high-quality CdSe needs to be made water-dispersible using small charged ligands (Chan and Nie, 1998), such as, mercaptopropionic acid or cystamine (Fig. 20.10). Charged surfactants can also be used that can intercalate with the hydrophobic ligands. QDs can also be dispersed in aqueous solution by attaching bulky polymeric surface ligands such as polyethylene glycol (Fig. 20.10). Electrostatically stabilized QDs are much smaller than sterically stabilized QDs but the former is more effective for biological applications as they can easily enter into the matrix but they aggregate in buffers of high ionic strength, whereas sterically stabilized QDs are less sensitive to ionic strength. Thus, the best possible way is to use smaller but still bulky charged ligands (Nann, 2005), such as polyethyleneimine or polyelectrolytes (Mattheakis et al., 2004), or an additional amphiphilic inorganic shell such as silica, which can further be functionalized (Figure 20.10). Attachment of organic dyes to biomolecules, such as, peptides, proteins, or oligonucleotides requires suitable functional groups for covalent binding. The advantage of organic dyes in this regard is that there is availability of functionalized dyes with established labelling protocols, purification and characterization techniques for dye bioconjugates (Waggoner, 2006). Further, small size of the organic dye minimizes steric hindrance and removes the possibility of interference with functions of biomolecules. Several fluorophores can be attached to a single biomolecule to maximize the fluorescence signal, but site-specificity is a problem with these dyes, also. Again, high-label densities can result in fluorescence quenching, depending on the structure of the dye, charge, and hydrophilicity (Mujumdar et al., 1993; Gruber et al., 2000). Excepting fluorescence quenching, QDs face similar challenges like the organic dyes. Till date there are no optimized methods for labeling biomolecules with QDs (Xing et al., 2007). The general principle is to make QDs water dispersible and then attaching them to biomolecules via electrostatically or covalent binding (Fig. 20.10). Moreover QDs have a tendency to aggregate if surface passivation is improper. When several biomolecules are tagged to a single QD (Goldman et al., 2004), then orientation of biomolecules become uncontrollable sometimes. This in turn may affect spectroscopic properties and colloidal stability of QDs and also functionality of biomolecules. Targeting cells may become difficult due to larger size of QDs. Hydrothermal synthesis of CdTe and their bioconjugation with Bovine Serum Albumin (BSA) (Li et al., 2007) is reported



Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants  863

Figure 20.10.  Overview of strategies to prepare water-dispersible QDs and QD bioconjugates. (a) QDs bearing hydrophobic ligands after preparation in organic solvent. HDA, hexadecylamine; TOPO, and trioctylphosphineoxide. (b) Ligand-exchange strategies to generate water-dispersible QDs. Illustrated are electrostatic colloidal stabilization (left), electrostatic and steric stabilization (middle), and steric stabilization of colloid (right). (c) Coupling of waterdispersible QDs to biomolecules, oligonucleotides. (d) Alternatively, the QDs bearing hydrophobic ligands can be subjected to direct ligand and exchange.

864  Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants

in a recent study where the crucial role of temperature has been stated in the colloidal synthesis of the QDs of various sizes keeping the pH and concentration ratio of ligand to monomer constant. The absorption spectrum of the bioconjugate differs from the standard absorption spectra of QDs and proteins, which confirms the successful conjugation of QDs with protein molecules. The microenvironment affects spectral position, fluorescence quantum yields, fluorescence lifetime of an organic fluorophore. The different parameters of microenvironment include polarity, proticity, viscosity, pH, ionic strength. The presence of surfactants, fluorescence quenchers like oxygen also affects the spectral and temporal properties of an organic dye acting as a label. In case of QDs, the microenvironment effect on the spectroscopic features is majorly governed by the accessibility of the core surface (Medintz et al., 2005). The QDs that are properly shelled with ligands are less sensitive to the microenvironment polarity provided that there is no ligand desorption (Ji et al., 2008). Emissions from QDs are not affected by the viscosity but with change in ionic strength electrostatically stabilized QDs tend to aggregate. Not many studies are available on the effect of the microenvironment on the spectroscopic properties of QDs as there is a large variety of the coatings used in QDs.

4  Quantum Dots: Sensing Foodborne Pathogens and Toxicants 4.1 Contaminants: Farm to Fork Public concern over food safety and food quality has gained immense attention over the past decade. Food can be contaminated in different stages and operations involved in production, processing, distribution, storage, and handling of a food and food ingredients from primary production to consumption. Monitoring contaminants (chemical or biological) in food is important as such contaminant affects human life and animal health, as well as international trade of agricultural products and food commodities. Industrial agricultural practices have evolved as a result of globalization; this, on one hand, has increased the food production but on the other hand also increased the potential likelihood of food contamination. There is a recent report that 31 major foodborne pathogens are the cause of sickness of 9.4 million people, 55,961 cases of hospitalizations, and 1,351 deaths every year in the United States (Schallan et al., 2011). The major cause of food contamination across globe is use of pesticides and herbicides.



Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants  865

Other major chemical contaminants like mycotoxins are worldwide contaminants of food and feed that cause health problems and economic losses. Biotoxins are toxic substances produced by and derived from plants and animals. Pesticides are substances or mixture of substances intended for preventing, destroying, repelling, or mitigating any pests or weeds. The enormous use of pesticides resulted in significant groundwater contamination due to their accumulation in the food chain, which gets filtered through various food formulations and drinking water. On the other hand the most common foodborne pathogens are Escherichia coli O157:H7, some strains of Staphylococcus aureus, Bacillus anthracis, Shigella spp,. Salmonella spp., Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, Listeria monocytogenes, Yersinis enterocolitica, Vibrio cholera, and Coxiella burnetti (Moss and Adams, 2008; Feng, 2001). In recent times, there are reports of several mycotoxin outbreaks that pose a serious threat to human and animal health. Lately pharmaceutical residues have become an increasing problem due to the use of antibiotics. They are easily transferred from the animals to humans and the effectiveness of antibiotics for common treatments gets highly diminished.

4.2 Sensing Methods Although there are several standard methods available for the detection and quantification of contaminants in food samples, but all those are laborious, time-consuming with limited specificity and sensitivity. Sensing utilizing QDs is one of the nondestructive methods that is quite effective for the detection of foodborne pathogens and toxins. For QDs to act as biosensors they need to be water dispersible, and so aqueous methods are preferred over other chemical synthesis methods. The concentrations of the precursors, the capping material, and the solvents are the primary factors for the colloidal synthesis of QDs. At considerably high temperatures, the precursors convert them to monomers and undergo supersaturation favoring nanocrystal growth through the process of nucleation. Temperature being one of the crucial factors in the growth of nanocrystals should be high enough such that it enables the rearrangement and annealing of atoms during the synthesis process. The smaller particles grow faster at high monomer concentrations, resulting in monodisperse particles, and the process is termed focusing. Colloidal synthesis results in highly monodisperse-size tunable nanomaterials with suitable optical properties for biosensor applications. Biocompatibility needs to be addressed in the first place, before conjugating QDs with biomolecules, as most of the biological reactions (except

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those that involve lipids) are hydrophilic in nature. In most of the cases, capping materials with a thiol group, such as, mercaptoacetic acid, thioglycerol, or mercaptoethanesulfonate, along with peptides that contain cysteine as the terminal amino acid group or having multiple cysteine groups are found suitable to be used as surfactants for the dispersal of QDs in aqueous solutions. A broad, well-defined absorption spectrum and a narrow emission band with full-width half-maxima (FWHM) of 50–60 nm for QDs are preferred for bioconjugation. Three basic methods are prevalent for conjugation of QDs to biomolecules. There can be possible cross-linking of the amine, carboxyl, or thiol group, which are available on the surface of the QDs, secondly directly attaching the biomolecules to the surface atoms of the QD through bonding of thiol groups or metals. The third method is via electrostatic interactions between the oppositely charged biomolecules and the QD surfaces. The method adopted for conjugation will depend upon the target biomolecule to be detected. Again each and every biomolecule have certain advantages and disadvantages for its use in specific targeting for detection of foodborne pathogens and toxins. The performance of each biomolecule for detecting a specific bacterial target is highly dependent on the complex food matrix and the growth of the bacteria itself. First, the food matrix is quite complex in nature; in addition to that, the different components present may cause possible degradation or nonspecific binding to the biomolecule, which will result in erroneous results. There are different biomolecules that can be attached to watersoluble QDs for specific biological targeting (Table 20.2) (Burris and Stewart, 2012), such as, nucleic acid, antibodies, carbohydrates, and so forth. The nucleic acid consisting of 15–25 nucleotides with a fluorescent molecule at one end and a quencher molecule at the other end can serve as a probe for sensing. In an unbound condition the fluorescence remains quenched due to the structural orientation as the fluorescent species reside in very close proximity to the quencher molecule. In presence of the target molecule the nucleic acid finds its complementary nucleic acid sequence, binds to it and fluorescence is switched on for possible detection. This methodology was first developed by Tyagi and Kramer (1996) and further modifications of this technology have led to new, improved methods for detection of foodborne pathogens in vitro using qPCR. Liming and Bhagwat (2004) detected single Salmonella species in fruits and vegetables using the aforementioned technique. Antibodies are also in frequent use as biomolecules coupled with QDs to act as detectors for foodborne pathogens and toxins. Monoclonal and Polyclonal are two types of antibodies used in



Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants  867

Table 20.2  QD-Based Sensors Coupling Biomolecules for Foodborne Pathogen Detection Sensor Type

Pathogen(s)

Target Detection Biomolecule Level

Food Matrix

Detection Time References

Nucleic acid

S. typhimurium

Nucleic acid

10 pM DNA

N/A

N/D

Vasudev et al. (2011)

E. coli O157:H7

Nucleic acid

9.72 fM DNA

N/A

N/D

Kim and Son (2010)

lmmunoassay E. coli O157:H7

Antibody

7.3 log CFU/ mL

N/A

N/D

Hahn et al. (2005)

E. coli O157:H7

Antibody

<10 CFU/mL

N/A

2h

Sanvicens et al. (2011)

E. coli O157:H7

Antibody

3 log CFU/mL

N/A

<2 h

Su and Li (2004)

E. coli O157:H7

Antibody

4 log CFU/mL

N/A

<2 h

Yang and Li (2006)

Antibody

5.5 log CFU/ mL

Milk (diluted)

<2 h

Zhao et al. (2009)

3 log CFU/mL

Apple juice <2 h

S. typhimurium S. typhimurium, S. flexneri, and E. coli O157:H7 Phage-based

E. coli

Protein

20 cells/mL

Water

<1 h

Edgar et al. (2006)

PCR

E. coli

Lectin

4 log CFU/mL

N/A

N/D

Mukhopadhyay et al. (2009)

Flow cytometry

E. coli O157:H7

Antibody

6 log CFU/mL

Water

N/D

Hahn et al. (2008)

Impedimetric

S. enteriditis

Antibody

Kim et al. (2007)

4 log CFU/mL

PBS

3 min

5 log CFU/mL

Milk

3 min

N/A, not applied to a food system; N/D, Not determined.

immunoassays. And monoclonal antibodies are found to be more specific than polyclonal antibodies on the basis of their action mechanism. Although there are certain disadvantages associated with the use of antibodies, their ability for simultaneous detection and separation of bacteria and toxins has kept them in active use as one of the tools for foodborne pathogen detection. Carbohydrates possess the capacity to bind with proteins and/or lipids, which enables them to detect pathogens and their toxins. The pathogens and toxins have the ability to recognize certain specific carbohydrate sequences and attach to their epithelial cells, this property of bacterial adhesion is utilized for the detection of pathogens and their toxins (Sharon, 2006). Unlike nucleic acids

868  Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants

and antibodies, carbohydrates have an affinity for widely different organisms, which gives these carbohydrates broad specificity. This is an added advantage for the carbohydrates as it enables them to target organisms that have gone though mutation and are different from the original organism. Furthermore, carbohydrates do not denature or lose their activity under extreme conditions like high temperature and/or pH changes, and this makes them appropriate to detect pathogens and toxins in food matrices. These QDs can be used in bioassays for detection of pesticides as these are highly photostable and the spectral properties of the QDs make them suitable for multianalyte detection. Nichkova et al. (2007) developed a multiplexed immunoassay using QDs for detection of atrazine mercapturate (AM) and 3-phenoxybenzoic acid (PBA). In case of pyrethroid insecticide and herbicide atrazine, PBA and AM serve as biomarkers. The working principle of these immonoassays is based on the immunointeractions between anti-PBA antibody and biotinylated anti-AM antibody and their corresponding antigens. Both the antigens, BSA-PBA and BSA-AM, are immobilized and are allowed to react with anti-PBA antibody and biotinylated anti-AM antibody, respectively. The above complexes are further incubated with antirabbit secondary antibodies that are conjugated to QD580 and streptavidin-QD620 and then successively were utilized to obtain fluorescence signals. In another study a rapid detection method for the herbicide 2,4-D was developed using CdTe QDs (Vinayaka et al., 2009). The quantitative estimation of the pesticides depends on the competitive binding of the free pesticide and a known concentration of QDbioconjugated pesticide toward the immobilized antibody in the immunoreactor column. There are considerable number of reports and studies where QDs have been potentially used for the detection of foodborne pathogens and their toxins in processed and unprocessed food and have gained importance in recent times. Bacterial toxins are of two types, endotoxins that cause lysis of the cell wall and exotoxins that cause disruption of normal cellular metabolism. Foodborne botulism occurs due to ingestion of botulinum toxin produced by the bacterium Clostridium botulinum. Warner et al. (2009) have designed and developed a fluorescence sandwich immunoassay for the detection of botulinum neurotoxin serotype A (BoNT/A). In this particular assay QDs and high affinity antibodies are utilized as reporter molecules. The antibodies bind to nonoverlapping epitopes (part of antigen) present in toxin and the recombinant fragment. The toxin gets quantified through the fluorescence obtained from QD. E. coli produces two types of toxins, heat-stable and heat-labile. The cell surface of E. coli contains



Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants  869

mannose-specific lectin. A carbohydrate functionalized CdS quantum dots was synthesized for the detection of E. coli (Mukhopadhyay et al., 2009). CdS QDs are coated with thiolated mannose that specifically can recognize the mannose-specific lectin in E. coli. Goldman et al. (2002) have proposed a multiplexed fluoroimmunoassay that is based on CdSe-ZnS core–shell QDs for the detection of cholera toxin (CT), Shiga-like toxin (SLT), ricin, and Staphylococcal enterotoxin (SEB). The detection of SEB in food products is crucial as it has the ability to cause toxic-shock syndrome. In order to attain specificity in the assay, toxin-specific antibodies were used as recognition molecules. Nonspecific interactions and background fluorescence from the QDs along with antigen–antibody cross-reactivity interfere with the assay. Interactions between individual QDs may cause spectral overlap and may interfere with the multiplexed analysis. A multicolor quantum dot nanoprobe was designed by Ho et al. (2005) for the detection of a pathogenic strain of Bacillus anthracis. The detection principle is suitably based on the identification of rpoB, which is a chromosomal gene marker and can differentiate Bacillus anthracis from other Bacillus species and also plasmid markers like pagA and capC known to determine anthrax toxin. Oligonucleotide probes known to be complementary to each gene marker are being designed specifically and conjugated to QDs with different emission wavelengths. The probes are also designed to form a sandwich nanoassembly that binds simultaneously to the target DNA molecule. The fluorescence signal obtained from each conjugated QD determines the presence of rpoB, pagA, or capC.

4.3 Toxicity and Future Potential of QDs The cytotoxicity of QDs in vivo studies is an important issue. Toxicity due to heavy metals due to the possible interactions of the QDs with cells should be taken care of before proceeding for vitro and vivo studies. The cytotoxicity that is due to cadmium present in many of these nanocrystals is well known. It is critical to recognize that whether these cytotoxic substances can leak out of the QD particles over time, upon illumination or oxidation (Zhang et al., 2006; Hoshino et al., 2004; Ma et al., 2006), in addition whether ligands or coatings are cytotoxic (Lewinski et al., 2008). QDs are chemically synthesized and the physicochemical properties and toxicity of these chemicals should be tested in organisms. There can be problem due to the posttreatment of the sample and QDs disposal when used as biosensors and that may subsequently lead to toxicological effects on the whole ecosystem. For in vivo applications, a biocompatibility factor needs to be addressed when QDs

870  Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants

are bioconjugated with adequate biomolecular capping materials like immortalized cell lines, liposomes and phospholipid vesicles. Biosensors based on nanofabrication are proving to be emerging tools for detection of food contaminants, such as, pesticide residues, foodborne pathogens, and their toxins. These sensors have additional advantages in terms of cost per assay, minimum sample volume, time taken for analysis, and potential for multiple-analyte analysis. Bioconjugated QDs are applied for multianalyte detection of DNA, proteins, pesticides, and so forth. Biosensors utilizing optical properties of QDs in nucleic acid assays can detect target DNA sequences, polymorphic regions, and gene expression. On the other hand, there is a considerable progress in the field of chemically modified QDs and their applications for optical sensing analytes like ions (cations or anions), monitoring pH changes. QD conjugates are implemented for different optical-sensing systems employing fluorescence, resonance energy transfer (FRET), and luminescence electron transfer (ET). QDs have a bright future in NIR fluorescence in vivo imaging, which requires labels that exhibit high fluorescence quantum yield in the 650–900 nm window, have adequate stability, good aqueous solubility, and low cytotoxicity in conjunction with large two-photon action cross-sections as desired for deep tissue imaging. Moreover QDs are attractive candidates for the development of multifunctional composite materials for the combination of two or more biomedical imaging modalities, like NIR fluorescence-magnetic resonance imaging. Although there is substantial achievement in the area of molecular sensing with QDs, still there are certain challenges. Till now, in most of the studies, the photophysical properties of QDs and electrical, optical, binding properties of the chemically modified layers are separately studied. In the QDs with core–shell nanostructures, the shell layer can actually participate in the binding or the activation of the analyte toward its interaction with QDs. Again, catalytic materials can be incorporated into the capping layer in order to activate the substrate analyte for its interaction with QD. QD-based detection system can bring revolution in the area of food safety and quality as they ensure rapid, reliable and sensitive detection of contaminants in food samples.

5 Conclusions In order to ensure safer production, distribution, and storage of food materials require cheaper, accurate, and robust chemical sensors. Fluorescent nanosensors are promising candidates for analyzing food contaminants, such as, pesticide residues, foodborne pathogens, and their toxins. However, most of such sensors



Chapter 20  Fluorescent nanosensors: rapid tool for detection of food contaminants  871

do not qualify the requirement of mass production. There is an array of inorganic and organic materials to synthesize quantum dots that possess posses interesting electrical and optical properties and chemo/stereo specificity. A continuous study of these sensors is going on to make them suitable for mass production using different synthetic routes and reaction engineering approach. Applications of nanosensors are also being focused. In addition to that a hurdle for usage of these sensors in commercial space is their toxicity issues. The synthetic particles due to their nanosize react very differently from their bulk counterpart both in physiological and in environmental condition. Hence, a considerable number of research projects are dedicated to toxicological evaluation for these nanosensors.

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