Water Soluble Fluorescent Nanocrystals Biology Essay

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Biomedical imaging and diagnostics rely heavily on efficient and reliable techniques of molecular imaging in biological systems [1]. This can be achieved by the development of novel and sophisticated probes such as nanoparticulate labels which have many advantages over conventional contrast agents such as increased contrast, lengthy circulation time and ease of integrating multiple properties [2][3].

The design of effective contrast agents requires careful consideration of the properties required for application. The contrast agents can be optimised by varying their surface coating, targeting properties, size, and degree of biocompatibility[4]-[6]. Quantum dots are excellent contrast agent for fluorescent imaging, with properties such as wide excitation windows, narrow emission windows, not prone to photo bleaching etc [7]. Until recently the limitation for the use of Quantum dots was that it was not water soluble.

Bawendi et al have devised a method of coating quantum dots with an outer layer thus rendering them water-soluble and also enhances the optical properties and robustness of nanocrystals in aqueous solutions [6]. Quantum yield between 10% -30% in water is observed in the photoluminescence of the coated quantum dots. Another advantage of this method being the coupling capacity ability of these water soluble quantum dots [6].

2. Nanotechnology in Biomedical Imaging

The fundamentals of basic and applied biological research rests upon the capacity to visually analyse bio molecules within living systems [8]. A number of fields such as genetics, molecular biology, medical diagnosis (e.g. imaging) and therapeutic purposes (e.g. targeted drug delivery) rely heavily on accurate imaging technologies [9][10]. A large number of which rely upon the phenomenon of fluorescence, thus giving rise techniques and instrument such as Confocal Microscopy, Epi Fluorescence Microscopy, Total Internal Reflection Fluorescence Microscopy (TIRF) etc [11]. The success of these sophisticated microscopes has led to an interest in developing new fluorescent probes which have an edge over conventionally used organic fluorophores [7]. Nanotechnology has fueled this interest by providing fluorescent probes (Fluorescent Quantum dots or Nano crystals) with novel properties that enhance the quality of images obtained (Fig.1) [12].

http://www.nano.gov/html/news/SpecialPapers/Quantum%20Dots%20for%20Fluorescence%20Imaging%20Reporter%20Roundtable%20061604_files/image002.gifhttp://www.nano.gov/html/news/SpecialPapers/Quantum%20Dots%20for%20Fluorescence%20Imaging%20Reporter%20Roundtable%20061604_files/image004.gif

Fig.1 Comparison of quantum dot fluorescence with conventional methods (A) In vivo imaging of vasculature of mouse with quantum dots (B) In vivo imaging of same area with conventional methods with five times the illuminating power [13]

2.1. Quantum dots as fluorescent labels

The characteristics of a good fluorescent label is that it is (i) conveniently excitable and detectable , (ii) it is bright (possess a high fluorescence quantum yield), (iii) biocompatible, (iv) stable under relevant conditions, (v) site-specific labelling can be achieved by addition of functional groups, (vi) suitable for multiplexing, (vii) low level of toxicity and (viii) reproducible [7][14]. In comparison to organic dyes, quantum dots possess many of these properties. Some of which are discussed below.

2.1.1. Optical properties

In contrast to organic fluorophores, quantum dots have a broader excitation spectra and a sharply defined emission spectra (Fig. 2), thus enabling excitation of multicolour quantum dots simultaneously without signal overlap by using a single light source [8].Due to quantum stark effect, the absorption and emission spectra of quantum dots can be adjusted according to the size of the quantum dot [9]. Also there is a large Stokes shift, which reduces autofluorescence and thus increasces sensitivity [9]. Due to the inorganic composition of Qds, they are stable and thus display decreased photobleaching compared to organic dyes [7].

Fig.2 The (a) absorption and (b) emission spectra of an organic dye (rhodamine) and QDs. The organic dye has a narrower absorption and emission spectra.a compared to quantum dotss [7].

Quantum dots with a narrow size distribution cab be manufactured by size selective precipitation of the crystallites from the growth solution, thus increasing the light emission in narrower spectral widths. This has been achieved by Bawendi and his co-workers [6]. Another attractive property of Quantum dot is that their fluorescence time is longer than that of organic dyes (fig. 3) [7].

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Fig.3 Comparison of fluorecent intenities of organic dyes( Nile red and Cy5) and quantum dots (CDSe/ZnS) [7].

The photoluminescent yield of the nanocrystals can be increased by reacting the surface of the nanocrystal with passivating ligands. The result of this being a sudden increase in the chemical potential at that surface. This thus eliminates the decrease in optical properties of the material by blocking the presence of traps for charge carriers. It has been seen that passivating quantum dots with an inorganic coating makes them tougher in comparison to quantum dots that are organically passivated [7].

2.1.2. Stability of fluorescent quantum dots

A contrast agent must be stable under different conditions such as in varying buffer media, cell environment etc . It should also not aggregate or precipitate under these varying conditions [7]. This property is dependent on the surface coating of the quantum dot. For eg. CdSe can be made water-dispersible by using charged surfactants or polymeric surface ligands.

2.1.3. Biocompatibility

In order for QDs to be used for biological application, it is essential that they be water soluble. Water soluble quantum dots have been prepared but they have their limitations, such as degradation over time and pH responsive nature [6][7]. Stable water soluble fluorescent nanocrystals have been developed by Bawendi et al [6], thus broadening the scope of these QDs for biological application.

2.1.4. Influence of microenvironment of contrast agent

The properties of fluorescent label vary according to the environment and temperature they are in. In the case of QDs, the microenvironment effect on spectroscopic features depends upon the type of ligand and strength of ligand binding (i.e. access to the core) Normally QDs are not very sensitive to microenvironment as compared to organic dye. Also, QD emission is hardly receptive to changes in viscosity, which is not the case in many organic dyes [7].

3. Water soluble fluorescent nanocrystals

Bawendi and his co-workers have developed water soluble fluorescent nanocrystals (QDs) that display all the above mentioned properties of a good fluorescent label [6][15]. Their work has thus resulted in water-soluble semiconductor nanocrystals that diplay high quantum yields with photoluminescence emissions and exhibit chemical and electronic stability in aqueous systems and has the added feature of coupling or linking capability.

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Fig. 4. Water soluble fluorescent nanocrystal depicting the nanocrystal (10), an overcoating layer (12), an outer layer (14) consisting of a compound (15) having a linking group (16) and one hydrophilic group (20) separated by a hydrophobic region (18) [6].

In 2002 an invention by Bawendi et al led to water-soluble fluorescent semiconductor nanocrystals comprising: a non-doped semiconductor material (made of either AlS, ALP, AlAs, AlSb, CdSe, GaN, CdTe, GaP, InAs, GaSb, InP, PbS, InSb, PbSe ZnS, GaAs, ZnSe, ZnTe, CdS, Ge, Si and ternary and quaternary mixtures of it), a layer overcoating the semiconductor material, and an outer layer . The overcoating layer consists either of the following: CdSe, CdS, GaN, ZnS, ZnSe and magnesium chalcogenides. The overcoating layer not being identical to the quantum dot and its band gap energy should be higher than the quantum dot. [6]

(b)

(a)

The outer layer consists of a compound having either one or more linking group for linking to the overcoating layer and one or more hydrophilic group .The overcoating layer and the linking group are separated by a hydrophobic region. The linking group of the outer layer comprises a moiety from amines, thiols, phosphines, phosphine oxides, and amine oxides .There can be one or more hydrophillic groups, one of which is a polar group which constitutes of either an alkoxide, ammonium salt, carboxylic acid, carboxylate, hydroxide, phosphate and sulfonate, which is cross linkable and the other unsaturated hydrophilic group constitutes either an acrylic acid, methacrylic acid or hydrophilically derivatised styrene. The hydrophobic region between the linking group and the hydrophilic group consists of an alkane, an alkene or other compounds capable of attractive interaction with each other, with a hydrocarbon chain of the formula --(CH.sub.2).sub.n --, where n ≥ 6, 8 or 10 and in which the hydrophobic regions of adjacent compounds are crosslinked [6].C:\Users\hanan\Desktop\6.tif C:\Users\hanan\Desktop\7.tif

Fig.5 (a) a water-soluble nanocrystal having cross linked hydrocarbon hydrophilic backbone, (b) a water-soluble nanocrystal constituting a polymethacrylate region [6].

The outer layer could also be a bilayer comprising an inner and outer layer. The inner layer comprises a lyophilic compound which is either of the following: an alkyl amine, trialkyl phosphine and phosphine oxide. Whereas the outer layer comprises a surfactant from the group consisting of sodium dioctyl sulfosuccinate, and a sodium salt of acids [6].

3.1. Method for the preparation of water-soluble nanocrystals

Initially nearly monodisperse nanocrystals are prepared by using a colloidal growth process employing a a high temperature, and subequently by size selective precipitation in order to obtain narrow spectral emission bandwidths. The appropriate overcoating layer is then coated upon the quantum dot and is prepared in such a way that the overcoating layer has a higher band gap as compared to the quantum dot. In order to produce water soluble nanocrystals, its surface is modified. This can be carried out by repetitive exposure to a competing coordinating group. Substitution of the water-solubilising compound does not have to be complete but rather enough to make the compound water soluble, which depends on the number of charged groups on the water-solubilising compound [6].

Fig. 6 Schematic representation of the formation of a water-soluble nanocrystal [6].

3.2. Factors that could hinder the widespread application of these water soluble fluorescent nanocrystals

The water-soluble layer of the invention by Bawendi et al has opened possibilities for producing nanocrystals with different combinations of nanocrystal core and over coating displaying improved robustness, photoluminescent emissions and stability in water-based solutions [6][15][16]. This is a leap in the field of biomedical imaging and diagnostics, but its reproducibility and toxicity are important factors to be considered [7].

3.2.1. Toxicity: While dealing with quantum dots, the cytotoxicity of elements such as cadmium (constituting the nanocrystal core) has to be considered [7][13]. It is of vital importance to evaluate the possibility of leakage of these cytotoxic elements out of the nanocrystal. Studies regarding a large number of different coatings and their cytotoxicity are being conducted, but research into this area is still ongoing [13]. Also, nanotoxicity of QDs could pose a problem in some cases but studies concerning most size of QD suggest otherwise [13].

3.2.2. Reproducibility: The requirement of fluorescent labels with reproducible physical and chemical properties is essential for reliable fluorescence measurements [7]. The broad range of surface functionalities possible makes the characterisation and study of quantum dot bioconjugates a challenge.

4. Conclusion

Optical properties of quantum dots are determined by the size distribution (dispersity), type of material and surface chemistry of the quantum dots. One of the major limiting factors for the widespread use of inorganic quantum dots a fluorescent labels was its inability to dissolve in aqueous solutions. Bawendi et al have overcome this difficulty by coating the quantum dot with suitable materials that render the nanocrystal water soluble. Their work has resulted in robust water-soluble nanocrystals that have display high quantum yields with spectral emissions of high purity that displays chemical and electronic stability in water and also can be coupled or linked.

Despite any disadvantages, the versatility quantum dots will prove to be of great importance , which is even capable of outweighing the disadvantages. Thus fluorescent nanocrystals or water soluble nanocrystals to be precise are a crucial part of the next generation of contrast agents for medical imaging.

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