Quantum Dots: The Biotags | Nanobiotechnology

Let us make an in-depth study of the quantum dots. The below given article will help you to  learn about the following things:- 1. Introduction to Quantum Dots 2. Surface Coatings of Quantum Dots 3. Size and Shape of Quantum Dots 4. Fluorescence Imaging of Bio­logical Samples 5. Live Cell Imaging: Passing the Plasma Membrane 6. In Vivo Imaging with Q-dots 7. The Cytotoxicity of Q-dot Probes and 8. Beyond Imaging 

Introduction to Quantum Dots:

Quantum dots (Q-dots) are nanometer-sized crystalline clusters (1-10 nm) that are syn­thesized from a variety of semiconductor materials. At such small scales, q-dots retain some of the bulk properties of the material from which they are derived, but also adopt new properties that directly depend on their size.

In term of photo-physics, this translates into a composition, shape and size dependent luminescence with absorption and emission bands that scale with the bulk band gap en­ergy of the material and the final diameter of the q-dot clusters.

Q-dots are characterized by large absorption spectra, but narrow and symmetric emission bands (full width at half maximum of 25-35 nm) that can span the light spectrum from the ultraviolet (UV) to the infrared (IR) (400-1,350 nm).

In addi­tion, they possess an excellent photo-stability (many orders of magnitude longer than con­ventional organic fluorophores) and quan­tum yield (the ratio of emitted to absorbed photons) as high as 90%. They also have large absorption cross-sections and long fluores­cence lifetimes (410 ns).

With all these fea­tures, q-dots have rapidly emerged as poten­tial new fluorescent probes for the imaging of biological samples. Indeed, they offer many advantages over conventional fluorophores for imaging techniques such as two-photon or time-gated microscopy, while allowing multicolor, long term and high sensitivity fluorescence bio-probes.

Surface Coatings of Quantum Dots:

The first demonstration of q-dots utilization for biomedical applications by Bruchez et al and Chan et al in 1998 have been followed by 2 years with very few publications using q-dots as bio-probes, eventually expanding exponen­tially after 2000. Beyond being a testimony to the pioneering nature of both articles, this lag time also stresses the difficulties that had to be overcome to efficiently use these new fluorescent probes for biological questions, particularly in live cells and in vivo imaging.

Indeed, the complexity of biological environ­ments imposes stringent conditions on the stability and efficacy of reporter probes. Dur­ing biochemical processes, molecular inter­actions rely on the conformational flexibility of biomolecules to attain electrostatic, hydro­phobic and steric matching of a substrate or ligand with its biological target.

Q-dots, which are relatively large and rigid spheres of inor­ganic material, might appear at first rather ill-suited when probing such shape-sensitive processes. The solution resides in interfacing one or more soft organic layers with the in­organic q-dots.

Over the past few years, scien­tists have rivalled in ingenuity in developing robust and versatile surface chemistries and providing biocompatible organic interfaces that:

(i) Solubilize and stabilize q-dots in bio­logical buffers,

(ii) Maintain, their original colloidal and photo-physical properties,

(iii) Keep their size relatively small and

(iv) Pro­vide reactive groups for subsequent conjugation to biomolecules.

The aqueous solubilisation of q-dots synthesized from hydrophobic solvents usually involves either shielding or exchange of surface hydrophobic ligands with amphiphilic ones. Both approaches have ad­vantages and inconveniences. Surface shield­ing chemistries—such as encapsulation in phospholipid micelles or coating with am­phiphilic polymers—retain the original hy­drophobic q-dot surface ligand and efficiently preserve the q-dot photo-physical properties.

In particular, the fluorescence quantum yield is minimally affected. These approaches are readily applicable to other Nano-materials presenting similar hydrophobic surfactants on their surface, but often result in q-dots with final sizes three to four times larger than the original particles, bringing them in same size range as the smallest fluorescent polystyrene microspheres (20 nm).

While large size is a lesser issue for in vitro applications, it might be detrimental for entry and specific interac­tions in crowded biological environments, as for instance in live cells and in vivo ap­plications. This problem was illustrated by researchers who showed that large size q-dots have difficulties to access neuronal synapses in hypocampal neurons.

In contrast, surface chemistries replacing the original surface ligands usually produce particles with smaller final diameter (8-15 nm for CdSe/ZnA particles originally 4-9 nm in diameter). For CdSe/ZnS q-dots in par­ticular, surface modifications often involve coordination of thiolated hydrophilic ligands on the q-dot ZnS layer.

Early approaches us­ing mono-thiol ligands are now known to result in q-dots with poor stability in biologi­cal buffers owing to the detachment over time of these ligands. More robust surface chemistries involve the use of dithiol ligands and further coating with engineered pro­teins, cross-linking of ligands after surface exchange in the case of silica or dendrimer coating and the use of polymers with multiple anchoring point to the q-dot surface such as oligomeric phosphine ligands or polycysteinyl peptides that enhanced stability.

With the notable exception of the phosphine ligand chemistry, exchange surface chemistries re­quire tailoring of the amphiphilic ligands for each new nanomaterial, making such ap­proaching much less general than shielding chemistries.

When trying to adapt specific surface exchange chemistry to other materi­als, surface coating with peptides is particu­larly interesting since they are amenable to molecular evolution. Peptide display libraries in bio-engineered phage or bacteria have in­deed been shown to be very powerful tools to rapidly screen and select unique peptide se­quences capable of binding to semiconductor or metallic materials.

With the appropriate combinatorial techniques (a peptide library can consist of more than a billion random sequences), the unique versatility of the 21 natural amino-acid peptide code can, in prin­ciple, be harvested via accelerated evolution to select peptide sequences that specifically bind to any type of Nano-materials.

Beyond screening peptides for binding and stabilizing of various semiconductor q-dots (Inp, CdTe, PbSe, etc.), one can also envision the same approach to select peptides enhancing the quantum yield or reducing the fluorescence intermittency of q-dots to improve their photo-physical properties. This very practical goal is supported by recent discovery that peptide coatings can lead to significant increase in quantum yield for CDSe q-dots with graded CdS/ZnS shells.

Whether based on ligand shielding or exchange, all q-dot surface chem­istries are designed to provide reactive groups such as amine (-NH₂), carboxyl (-COOH) or Mercator (-SH) groups for direct conjugation to biomolecules. A growing set of functions (streptavidin, protein A, biotin, etc.) is avail­able for easy and customized conjugation to nearly all biomolecules of interest. ‘Ready-to- use’ q-dots equipped with those functions or with antibodies can now be purchased from different commercial sources.

With the pep-tide-coating approach, it is easy to construct a sequence that will present any of the pre­viously cited reactive groups at the surface. For instance, researchers have recently intro­duced thiol derivatized q-dots.

These q-dots are coated with peptides presenting a ter­minal cysteine, and are efficiently modified with biocytinmaleimide or used to conjugate full-length antibodies by reaction with heterobifunctional cross-linking reagents such as succinimidyl-4-(N-maleimidomethyl) cyclo- hexane-1-carboxylate (SMCC).

An alternative to cross-linking q-dots to full length proteins is to derivative them with polypeptides sequences capable of folding into active binding domains such as Src homology 2 (SH2) domains (domain composed of a beta sheet surrounded by two alpha helices that binds specifically to peptides containing phosphotyrosines) or PDZ domains (a beta-sandwich with two alpha-helices involved in high affinity binding at the c-terminal resi­dues of trans-membrane receptors and ion channels) which are involved in intracellular signaling.

Polycysteinyl peptides with more than 50 amino acids can be attached on the surface of q-dots, and application to small pro­teins domains, which are about 100 amino acids long, should provide q-dots with target­ing and binding functions while keeping their size relatively small.

Size and Shape of Quantum Dots:

Owing to their structure and size, q-dots have a large surface to volume ratio and, therefore, present a large number of surface attachment points that can be exploited to engineer mul­tifunctional q-dot probes. A common example of useful function demonstrated with nearly all published q-dot solubilisation chemistries is the incorporation of polyethylene gly­col (PEG) molecules in the amphiphilic or­ganic surface layer.

PEG not only enhances the aqueous solubility of the q-dots but also reduces nonspecific adhesion to biological cells. The non-toxic and excellent solvating properties of PEG polymers have previously been employed in drug development to im­prove bio-distribution and circulation time in vivo and to limit immunogenicity.

Pegylation has proven to be fully compatible with q-dot surface chemistries and is bound to play a prevalent role when optimizing in vivo pharmacokinetics of q-dot bio-probes. Combining multiple functions on q-dot surfaces could also be advantageous in different situa­tions. An example is in vivo targeting of can­cer cells, where this could help create probes with enhanced targeting selectivity. Tumor cells often over-express different cell surface markers that are usually absent or found only in low copy numbers in normal cells.

The large surface area of q-dots may be used as a scaffold to graft multiple ligands specific for these over-expressed markers. Assum­ing that these markers co-localize on the cell membrane (or are present at a large enough density), the q-dot probes should exhibit a large avidity for the co-localized target mark­ers and, therefore, have an enhanced relative affinity for tumor cells versus normal cells.

Non-specific binding to healthy tissue could be further reduced if low affinity ligands are chosen. Multi-potent probes might also prove useful to develop multi-contrast imag­ing as was recently demonstrated with in vivo positron tomography (PET) and fluorescence imaging using q-dots labeled with radioactive peptides.

On the other hand, the presence of mul­tiple reactive groups on the surface of q-dots can be problematic when trying to achieve a one-to-one q-dot-to-ligand ratio. Multiple ligands per q-dot can indeed result in unwanted side-effects such as target aggregation. This can interfere with many biological processes where molecules are activated upon mutual cross-linking by ligands.

In such cases, con­trolling the number of ligand per q-dots be­comes absolutely necessary to avoid activa­tion upon binding and to study biomolecules in their ‘resting’ state. Although highly desir­able, single-ligand derivatization of q-dots has not yet been achieved.

Fluorescence Imaging of Bio­logical Samples:

With a host of surface chemistries and the availability of commercial q-dots, biomedi­cal applications using q-dots have flourished. The multitude of successful uses in immuno­fluorescence assays, biotechnology detection, live cell imaging, single-molecule biophysics or in vivo animal imaging is a testimony of the great excitement generated by these new fluorescent probes and their tremendous po­tential to revolutionize fluorescence imaging techniques.

Excellent reviews on the subject have recently been published on the current applications of q-dot as bio-probes. The com­bination of a large absorption cross-section (extinction coefficient), good quantum yield and large saturation intensity makes q-dots brighter than fluorescence dyes or fluores­cent proteins.

The sensitivity of detection in fluorescence-based assays is, therefore, sig­nificantly enhanced. However, one should keep in mind that q-dots are bulky probes and, when bound to a target, a q-dot might prevent the access of another q-dot to a neighboring target molecule due to steric hindrance. For lambing of molecules expressed in high copy numbers and relive localized, this may result in a decrease of the total number of labeled molecules, compared to a staining performed with smaller fluorophores, and lead to a decrease of the over signal inten­sity.

Q-dots brightness is, therefore, best used when high sensitive detection of low copy numbers of molecules is desired, or when the molecules to be detected are sparsely distributed. In particular, q-dots are excellent probes for single-molecule fluorescence microscopy, which is one of the most exciting new appli­cations offered to biologists.

This was well- demonstrated by scientists who followed the lateral dynamics of q-dot-labeled single gly­cine receptors in the membrane of neurons. Taking advantages of the brightness and high photo-stability of the probes they were able to observe the diffusion and entry of glycine receptors in neuronal synapses with a spatial resolution of 5-10 nm.

With q-dot probes, bi­ologists equipped with a standard epifluorescence microscope and a good CCD camera can easily perform single-molecule imaging and use this powerful technique to detect biological events usually hidden in ensemble measurements or are inaccessible because of the fast photo bleaching of conventional or­ganic fluorophores.

Q-dots are orders of magnitudes more photo-stable than dyes. High fluorescence photo- stability allows immuno stained samples to be repeatedly imaged and crisp high resolution three-dimensional images to be acquired.

Resistance to photo bleaching becomes espe­cially advantageous in live cell experiments, where whole cells or even molecules can be observed and tracked for hours or days. Re­searchers convincingly demonstrated cell lineage tracing by injecting q-dots in a single Xenopus frog cell at an early embryonic stage and by following daughter cells over days. In marked contrast with fluosophores, q-dots have broadband photon absorption spectrum and narrow, symmetrical and tunable emis­sion bands which facilitate the simultaneous detection of multiple signals.

To date, q-dots emitting in the visible part of the spectrum (e.g. CdSe/ZnS) have been the most com­monly used. Highly optimized chemical syntheses for these semiconductor materials have made the production of these q-dots fairly easy and safe. CdSe q-dots’ range of emission wave­length matches perfectly the detection range of typical imaging and fluorescence devices (such as CCD cameras and photomultiplier tubes). With the extension of q-dot probes to new biological applications, the demand for new types of semiconductor materials has grown.

Significant advances have been made in the production and solubilisation of q-dots emitting in the near infra-red (NIR) and IR spectral range (CDTe/CdSe, InAs, etc.). Cur­rently, the available palette of semiconductor materials allows the synthesis of q-dot probes with emission wavelength ranging from 400 nm to several micro-meters.

Their broad ex­citation spectrum al­lows a single excitation wavelength to simul­taneously excite all q-dots emitting from UV to the IR. Multicolor imaging of biological samples is, therefore, significantly simplified, and improved multicolor co-localization stud­ies are made possible with the elimination of many chromatic aberration and alignment is­sues encountered with standard fluorophore microscopy.

A recent example of dynamic multicolor imaging, made possible by the long-term stability of q-dots is the interest­ing study of time-dependent co-localization of cell surface receptors involved in signal transduction. The narrow and tunable emis­sion spectra of q-dots have also recently been exploited to customized donor emission in fluorescence resonance energy transfer (FRET) assays between q-dot donors and flu­orescent dye acceptors.

FRET measurements with q-dots are able to convey good qualitative information about molecular associations in ensemble measurements and appear to have great potential as Nano scale biosensors. How­ever, there are still some problems to solve to be able to extract accurate distance measure­ments by FRET, as would be needed to study dynamic changes in the conformation of biomolecules.

One problem stems from the fact that, unlike chromospheres, q-dots from a single synthesis batch are structurally and spectrally slightly different from one anoth­er. This is due to the large number of atoms q-dots are made of, and to the various steps involved in their synthesis which can result in small variations and defects in the core, shell, or the biocompatible organic lay­ers.

These defects have significant influences on the q-dot photo physical properties. This heterogeneity would translate into a corre­sponding heterogeneity of the Forster radius R0, which would affect the precision of single molecule FRET measurements using q-dots, unless the actual spectrum of each individual q-dot can be measured.

Another potentially annoying characteristic of q-dot is their environment-dependent fluo­rescence intermittency, also known as blink­ing. Q-dot blinking is associated to charge trapping and un-trapping at surface defects during excitation and results in an alternation (at all timescales) of bright and dark states during which no photons are emitted.

Q-dot blinking, therefore, results in the random loss of distance information at all-time scales, preventing a recovery of the complete con­formational dynamics of the observed single molecule. In addition, blinking is strongly correlated with spectral jumping (change in the emission peak position) that can affect the FRET efficiency.

An intimate understanding and control of the photo-physic is, therefore, required to harvest the full potential of q-dots as FRET probes. Improvements such as the near-elimination of blinking in the presence of P-mercaptoethanol or dithiothreitol have been reported. Although live cells imaging at the high concentration of b-mercaptoethanol used (1 mM) might not be possible, this is already a tremendous amelioration for FRET applications in vitro.

Time-gated and lifetime imaging of biolog­ical samples labeled with q-dot probes are two other promising techniques taking advantage of the long fluorescence lifetime of q-dots. Q-dots have a fluorescence lifetime (time of fluorescence emission decay after excitation) of 20-30 ns that is significantly longer than the ubiquitous short-lived auto-fluorescence of cells (2 ns) or organic fluorophores life­times (1-4 ns).

Auto-fluorescence is the most common source of background and reduces detection sensitivity. The fluorescence decay of q-dots is long enough so that, by the time the auto-fluorescence signal of a specimen has vanished, q-dots still emit photons.

In time-gated imaging, early incoming photons are discarded to filter-out the auto-fluorescence signal and only retain the q-dot signal. This results in a dramatic contrast enhancement for cellular imaging. In a similar approach, fluorescence lifetime imaging microscopy can exploit differences in fluorescence decay rates to discriminate q-dot signal from that of other fluorophores within the same speci­men. The combination of multi-lifetime and multicolor imaging of fluorescence dyes with q-dot probes offers yet unprecedented multi­plexing capabilities for fluorescence imaging of biological samples.

Live Cell Imaging: Passing the Plasma Membrane:

Most live cells studies with q-dots have, un­derstandably, focused on membrane mark­ers since they are easy to access and do not require passage of the probes through the highly regulated and organized cell mem­brane. The obvious (but difficult) next step is to extend targeting to cytoplasmic molecules. Although some interesting attempts have been performed using membrane transloca­tion peptides, electroporation or transfection reagents, q-dots often tend to accumulate in vesicles or appear non-homogeneously dis­tributed in the cytoplasm.

As of today, there seems to be no real success to overcome this complication and the best technique for cy­toplasmic translocation of q-dots is the direct injection in living cells. This approach has al­lowed the targeting of q-dots to sub-cellular compartments such as mitochondria or the nucleus using targeting peptides.

Cell injec­tion, albeit useful for single-cell observation, is very time consuming when large number of cells are to be labeled. New methods to homogeneously distribute q-dots in the cyto­plasm of cells would, therefore, be welcome.

In Vivo Imaging with Q-dots:

Applications of q-dot probes to animal imag­ing have been surprisingly rapid, sometime overlooking fundamental yet unsolved is­sues such as cytotoxicity. Nonetheless, the results are spectacular. Q-dots have allowed high-sensitivity and high-contrast imaging in deep tissues in vivo not only in mice but also in larger species.

Intravenous injection in mice was performed to image blood ves­sels, to target tissue-specific vascular mark­ers, or to image lymph nodes. The first tar­geting of grafted tumors in vivo was also demonstrated with PEG and antibody-coated q-dots.

All these experiments advocate the use of PEG surface ligands to enhance the circu­lation time, reduce the dosage and improve the targeting specificity of q-dots in vivo. In one case, q-dots could be found in the bone marrow and the lymph nodes of mice several months after injection.

While researchers had to resort to spectral un-mixing algorithms to identify the proper location of targeted q-dots emitting in the vis­ible spectrum, another group of scientists in­jected NIR CdTe/CdSe q-dots emitting at 850 nm to perform nearly background-free imag­ing of lymph nodes 1 cm deep in tissues. The significant advances made in the production and solubilisation of NIR and IR emitting q-dots are particularly exciting for deep tissue imaging in vivo.

Combining these approach­es with time-gated microscopy to separate short auto-fluorescence lifetime signals of the animal from that of the much longer lifetime of q-dots could result in detection sensitivities approaching that of radio-labeled probes.

The Cytotoxicity of Q-dot Probes:

As the range of biomedical applications with q-dots expands to measurements in vivo, le­gitimate questions concerning their short and long term cytoxicity have been raised. Indeed, the composition of cadmium chalcogenide q-dot probes poses potentially serious health risks that should not be overlooked.

Metal cadmium, like the very potent ions of mercury and lead, is strongly poisonous. Cadmium has a half-life of about 20 years in humans and is a suspected carcinogen that can accumulate in the liver and kidney and bio-distributes in all tissues since there are no known active mechanisms to excrete cadmi­um from the human body.

Even if cadmium is present in q-dots in an inorganic crystalline form, possible toxicity by leakage of these ions following chemical degradation (e.g. in cell endosomes) or photo-degradation (e.g. during excitation) is of serious concern.

Un­fortunately, there have been a very limited number of studies specially designed to assess thoroughly the toxicity of q-dots, and most of the reports describing the potential toxic ef­fects of q-dots have used non-standardized methods and have often been performed by scientists that are not from the fields of toxi­cology or health science.

In addition, assessing the potential toxicity of q-dot probes appears to be relatively com­plicated matter since no q-dots are similar and different composition, coating and solubilisation chemistries might display different tox­icities. It seems fair to assume that q-dot toxic­ity will depend on multiple physical, chemical and environmental factors, such as q-dot size, charge, surface coating, concentration, chem­ical composition, colloidal and chemical sta­bility, production of free radicals or singlet oxygen upon excitation and exposition route, all of which might be determining factors of q-dot short and long term toxic effects in vivo.

Early reports using q-dots have not found sig­nificantly detrimental effects on the normal function or the morphology of cells, at least within the short time frame of the performed experiments. However, recent reports have in­dicated that bare CdSe q-dots are indeed toxic to cells through the release of free cadmium ions. While over coating of CdSe cores with a protective ZnS shell decreases cytotoxicity, the nature of the amphiphilic surface organic coating itself appears to critically influence the toxic properties of the particle.

In this re­spect, natural biocompatible surface coatings for q-dots such as peptides or sugars might be less nocuous to cells than other approaches. Nevertheless, lack of morphological change for cells is not a sufficient parameter to assess toxicity. Scientists recently reported that in human bone marrow mesenchyme stem cells the expression of specific genes is suppressed af­ter transfection of q-dots, despite no observable changes in cell proliferation.

Although the jury is still out to determine if q-dots are safe probes, a range of concentrations exists at which potential interference with nor­mal physiological processes can be reduced. Improving the detection efficiency and the brightness of q-dots will allow the use of lower concentrations and reduce potential cytotoxic risks.

As discussed above, the use of NIR emitting q-dots is very advantageous since their high visibility in vivo requires lesser material. As recently reported only 400 pmol of CdTe/CdSe q-dots were injected for the in vivo imaging of lymph nodes in a pig. This amount is far below the dose known to induce cadmium poisoning in animals.

Non- heavy metal semiconductor materials such as InGaP q-dots also offer an interesting alterna­tive with respect to cytotoxicity issues. The potential cytotoxicity of q-dots could, on the other hand, be of interest when employing them as therapeutic agents—for example in the destruction of cancer cells.

Several groups have suggested that not only cadmium ion release, but also free radi­cals and singlet-oxygen production by q-dots upon UV excitation might be harvested for photodynamic therapy.

Although much work still needs to be done in this domain, band- gap engineering of q-dots and development of thin surface coatings to optimize direct ener­gy transfer to oxygen or to surface conjugated photosensitizing agents is a highly exciting avenue to explore.

Beyond Imaging:

Although they are mainly employed for fluo­rescence imaging applications, q-dots have a yet untapped potential in bioelectronics. In­deed, beyond their exceptional photo physical properties, q-dots have also unique electronic properties that arise from quantum confine­ment in such small semiconductor clusters. Many efforts have been launched to interface q-dots with neurons, in an attempt to trig­ger neuronal functions by q-dot-mediated stimulation.

Although no clear proof of the feasibility of such projects has yet emerged, a thin and carefully designed surface coating together with q-dots optimized for large sepa­ration of charges after excitation (e.g. type II CdTe/CdSe q-dots) might indeed open the doors to a new generation of light-actable q-dot probes, where photo-generated carri­ers (electrons or hole) may undergo electron transfer, produce heat or generate dipole moments sufficiently strong to interact with membrane proteins and trigger changes in lo­cal membrane potentials.

The various surface chemistries developed over the years to interface ‘soft’ biological materi­als with inorganic q-dots have not only been essential for the successful use of q-dots in bi­ological imaging, but have also provided new tools in materials science for the controlled assembly of Nano-materials.

Indeed, these interfaces, when used as molecular glue be­tween inorganic Nano-scaled building blocks, offer means to control 2D and 3D assemblies of Nano-scaled objects by taking advantage of the unmatched ability of biological molecules to spatially and dynamically self-assemble and self-organize into complex molecular su­perstructures.

Programmable nanomaterial scaffolding, driven by self-assembling pep­tides, protein domains, DNA hybridization or carbohydrate/carbohydrate recognition, hold great promises for the creation and emer­gence of new biomaterials and the expansion of biomimetic nanotechnology.

In summary, within a few years, q-dots have recognition as very versatile bio-probes for fluorescence imaging in vitro and in vivo. They are available in a large assortment of emission wavelengths and in a variety of sur­face chemistries and conjugation strategies that permit derivatization with virtually any biologically active molecule.

While they are not the perfect fluorophores and more needs to be done to better understand and improve their photo-physics and to further assess their cytotoxicity—q-dots have found their place in the existing repertory of bio-imaging tools. They will, without doubt, be a probe of choice for long term, high-sensitivity and multi-contrast imaging of molecular dynamics in bio­logical samples.