In vitro and in vivo imaging with quantum dots

Quantum dots (QDs), also named semiconductor nanocrystals, have initiated a new realm of bioscience by combining nanomaterials with biology, which will profoundly influence future biological and biomedical research. In this review, we describe the extraordinary optical properties of QDs and developments in methods for their synthesis. We focus on fluorescent imaging with QDs both in vitro and in vivo, and the cytotoxicity of QDs and potential barriers to their use in practical biomedical applications. Finally, we provide insights into improvements aimed at decreasing the cytotoxicity of QDs and the future outlook of QD applications in biomedical fields.

Use of quantum dots in the development of assays for cancer biomarkers

Biomarker assays may be useful for screening and diagnosis of cancer if a set of molecular markers can be quantified and statistically differentiated between cancerous cells and healthy cells. Markers of disease are often present at very low concentrations, so methods capable of low detection limits are required. Quantum dots (QDs) are nanoparticles that are emerging as promising probes for ultrasensitive detection of cancer biomarkers. QDs attached to antibodies, aptamers, oligonucleotides, or peptides can be used to target cancer markers. Their fluorescent properties have enabled QDs to be used as labels for in-vitro assays to quantify biomarkers, and they have been investigated as in-vivo imaging agents. QDs can be used as donors in assays involving fluorescence resonance energy transfer (FRET), or as acceptors in bioluminescence resonance energy transfer (BRET). The nanoparticles are also capable of electrochemical detection and are potentially useful for “lab-on-a-chip” applications. Recent developments in silicon QDs, non-blinking QDs, and QDs with reduced-size and controlled-valence further make these QDs bioanalytically attractive because of their low toxicity, biocompatibility, high quantum yields, and diverse surface modification flexibility. The potential of multiplexed sensing using QDs with different wavelengths of emission is promising for simultaneous detection of multiple biomarkers of disease.

Preparation of liposomes loaded with quantum dots, fluorescence resonance energy transfer studies, and near-infrared in-vivo imaging of mouse tissue

We report on a simple, fast and convenient method to engineer lipid vesicles loaded with quantum dots (QDs) by incorporating QDs into a vesicle-type of lipid bilayer using a phase transfer reagent. Hydrophilic CdTe QDs and near-infrared (NIR) QDs of type CdHgTe were incorporated into liposomes by transferring the QDs from an aqueous solution into chloroform by addition of a surfactant. The QD-loaded liposomes display bright fluorescence, and the incorporation of the QDs into the lipid bilayer leads to enhanced storage stability and reduced sensitivity to UV irradiation. The liposomes containing the QD were applied to label living cells and to image mouse tissue in-vivo using a confocal laser scanning microscope, while NIR images of mouse tissue were acquired with an NIR fluorescence imaging system. We also report on the fluorescence resonance energy transfer (FRET) that occurs between the CdTe QDs (the donor) and the CdHgTe QDs (the acceptor), both contained in liposomes. Based on these data, this NIR FRET system shows promise as a tool that may be used to study the release of drug-loaded liposomes and their in vivo distribution.

CdS quantum dots as a fluorescent sensing platform for nucleic acid detection

We demonstrate that CdS quantum dots (QDs) can be applied to fluorescence-enhanced detection of nucleic acids in a two-step protocol. In step one, a fluorescently labeled single-stranded DNA probe is adsorbed on the QDs to quench its luminescence. In step two, the hybridization of the probe with its target ssDNA produces a double-stranded DNA which detaches from the QD. This, in turn, leads to the recovery of the fluorescence of the label. The lower detection limit of the assay is as low as 1?nM. The scheme (that was applied to detect a target DNA related to the HIV) is simple and can differentiate between perfectly complementary targets and mismatches.

Design of sugar–oligonucleotide conjugates installed gold nanoparticle for effective delivery to hepatic parenchymal cells

A novel oligonucleotide delivery system that is based on oligonucleotide–nanoparticle conjugates has been described. Installed oligonucleotides were modified with the carbohydrate at the 3′ terminus, accordingly, constructed nanoparticles display clustered carbohydrates on their outer layer for the targeted delivery of oligonucleotides. The method for the construction of ligand-functionalized nanoparticle was simple and reproducible. The stability of the nanoparticles displaying clustered carbohydrates greatly increased in serum compared to nanoparticles without carbohydrates. In order to investigate the targetability of oligonucleotide–nanoparticle conjugates into primary hepatic parenchymal cells, freshly isolated rat hepatocytes were incubated with nanoparticles and the amount of internalized gold nanoparticles was evaluated by an inductively coupled plasma mass spectroscopy analysis. Nanoparticles displaying clustered carbohydrates internalized more efficiently than nanoparticles without carbohydrate modifications. In particular, the cellular uptakes of oligonucleotide-conjugated gold nanoparticle increased 1.7 ～2.0-fold by galactose modification. Competition assay revealed that clustered galactose enhanced the internalization of the nanoparticle into primary hepatic parenchymal cells by a receptor-mediated process.

Quantum dots and polymer hybrid composites: new insights into fluorescence switch and turn-on anion sensing

We present a previously unexplored mechanism for a quantum dot (QD) fluorescence switch, and introduce its application to turn-on sensing of anions. A hybrid composite is formed from polyacrylic acid (PAA) and the surface of CdTe QDs through strong coordination interactions between the carboxy groups of PAA and the Cd atoms on the surface of the QDs. Such interactions cause almost complete quenching of the fluorescence of the QDs via the pull effect of PAA. Carbonate, silicate or phosphate anions are then added in the pH 8.2 solution of the QD-PAA composites. These anions are partially hydrolyzed and protonated, and then react with the coordinating oxygen atoms due to the formation of hydrogen bonds. This leads to a decrease in the pull effect of PAA, and eventually gives rise to a strong recovery of fluorescence. The detection limits are 0.29, 0.02, and 0.76 mM, respectively, for carbonate, silicate and phosphate. The surface modulation method presented here provides a novel strategy for the design of QD-based chemosensors for carbonate, silicate or phosphate which otherwise are difficult to detect.

Structural insights into calmodulin/adenylyl cyclase 8 interaction

Calmodulin (CaM) is a highly conserved intracellular Ca²⁺-binding protein that exerts important functions in many cellular processes. Prominent examples of CaM-regulated proteins are adenylyl cyclases (ACs), which synthesize cAMP as a central second messenger. The interaction of ACs with CaM represents the link between Ca²⁺-signaling and cAMP-signaling pathways. Thereby, different AC isoforms stimulated by CaM, comprise diverse mechanisms of regulation by the Ca²⁺ sensor. To extend the structural information about the detailed mechanisms underlying the regulation of AC8 by CaM, we employed an integrated approach combining chemical cross-linking and mass spectrometry with two peptides representing the CaM-binding regions of AC8. These experiments reveal that the structures of CaM/AC8 peptide complexes are similar to that of the CaM/skeletal muscle myosin light chain kinase peptide complex where CaM is collapsed around the target peptide that binds to CaM in an antiparallel orientation. Cross-linking experiments were complemented by investigating the binding of AC8 peptides to CaM thermodynamically with isothermal titration calorimetry. There were no hints on a complex, in which both AC8 peptides bind simultaneously to CaM, refining our current understanding of the interaction between CaM and AC8.

Fluorescence energy transfer-based multiplexed hybridization assay using gold nanoparticles and quantum dot conjugates on photonic crystal beads

A multiplexed assay strategy was developed for the detection of nucleic acid hybridization. It is based on fluorescence resonance energy transfer (FRET) between gold nanoparticles (AuNPs) and multi-sized quantum dots (QDs) deposited on the surface of silica photonic crystal beads (SPCBs). The SPCBs were first coated with a three-layer primer film formed by the alternating adsorption of poly(allylamine hydrochloride) and poly(sodium 4-styrensulfonate). Probe DNA sequences were then covalently attached to the carboxy groups at the surface of the QD-coated SPCBs. On addition of DNA-AuNPs and hybridization, the fluorescence of the donor QDs is quenched because of the close proximity of the AuNPs. However, the addition of target DNA causes a recovery of the fluorescence of the QD-coated SPCBs, thus enabling the quantitative assay of hybridized DNA. Compared to fluorescent dyes acting as acceptors, the use of AuNPs results in much higher quenching efficiency. The multiplexed assay displays a wide linear range, high sensitivity, and very little cross-reactivity. This work, where such SPCBs are used for the first time in a FRET assay, is deemed to present a new and viable approach towards high-throughput multiplexed gene assays.

Quantum dots on electrodes—new tools for bioelectroanalysis

The review covers recent developments in which quantum dots (QDs) are combined with electrodes for detection of analytes. Special focus will be on the generation of photocurrents and the possibility of spatially resolved, light-directed analysis. Different modes for combining biochemical reactions with QDs will be discussed. Other applications involve the use of QDs as labels in binding analysis. Different methods have been developed for read-out. In addition to photocurrent analysis, voltammetric detection of metals and electrochemiluminescence (ECL) can be used. In the latter, light is the sensor signal. ECL-based systems combine the advantage of very sensitive analytical detection with rather simple instrumentation.

Temperature-assisted ionic liquid dispersive liquid-liquid microextraction combined with high performance liquid chromatography for the determination of PCBs and PBDEs in water and urine samples

We report on silver–gold core-shell nanostructures that contain Methylene Blue (MB) at the gold–silver interface. They can be used as reporter molecules in surface-enhanced Raman scattering (SERS) labels. The labels are stable and have strong SERS activity. TEM imaging revealed that these nanoparticles display bright and dark stripe structures. In addition, these labels can act as probes that can be detected and imaged through the specific Raman signatures of the reporters. We show that such SERS probes can identify cellular structures due to enhanced Raman spectra of intrinsic cellular molecules measured in the local optical fields of the core-shell nanostructures. They also provide structural information on the cellular environment as demonstrated for these nanoparticles as new SERS-active and biocompatible substrates for imaging of live cells.

Spectral Reproducibility and Quantification of Peptides in MALDI of Samples Prepared by Micro-Spotting

Previously, we reported that MALDI spectra of peptides became reproducible when temperature was kept constant. Linear calibration curves derived from such spectral data could be used for quantification. Homogeneity of samples was one of the requirements. Among the three popular matrices used in peptide MALDI [i.e., α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and sinapinic acid (SA)], homogeneous samples could be prepared by conventional means only for CHCA. In this work, we showed that sample preparation by micro-spotting improved the homogeneity for all three cases.

Peptide–Column Interactions and Their Influence on Back Exchange Rates in Hydrogen/Deuterium Exchange-MS

Hydrogen/deuterium exchange (HDX) methods generate useful information on protein structure and dynamics, ideally at the individual residue level. Most MS-based HDX methods involve a rapid proteolytic digestion followed by LC/MS analysis, with exchange kinetics monitored at the peptide level. Localizing specific sites of HDX is usually restricted to a resolution the size of the host peptide because gas-phase processes can scramble deuterium throughout the peptide. Subtractive methods may improve resolution, where deuterium levels of overlapping and nested peptides are used in a subtractive manner to localize exchange to smaller segments. In this study, we explore the underlying assumption of the subtractive method, namely, that the measured back exchange kinetics of a given residue is independent of its host peptide. Using a series of deuterated peptides, we show that secondary structure can be partially retained under quenched conditions, and that interactions between peptides and reversed-phase LC columns may both accelerate and decelerate residue HDX, depending upon peptide sequence and length. Secondary structure is induced through column interactions in peptides with a solution-phase propensity for structure, which has the effect of slowing HDX rates relative to predicted random coil values. Conversely, column interactions can orient random-coil peptide conformers to accelerate HDX, the degree to which correlates with peptide charge in solution, and which can be reversed by using stronger ion pairing reagents. The dependency of these effects on sequence and length suggest that subtractive methods for improving structural resolution in HDX-MS will not offer a straightforward solution for increasing exchange site resolution.

A new label-free approach for the determination of reaction rates in oxidative footprinting experiments

Hydroxyl radical-mediated oxidative footprinting coupled to mass spectrometric analysis is an attractive technique for protein surface mapping, conformational changes monitoring, and protein–ligand interfaces mapping in solution. In this technique, a protein is oxidized by in situ-generated hydroxy radicals and the site and rate of oxidation can be determined by proteolysis followed by mass spectrometric analysis. Changes in peptide oxidation rate can then be correlated to the changes in solvent exposure, and information about conformational changes or interaction domains can be obtained. The method relies, therefore, on the accurate measurements of peptide oxidation rate. Here, we describe a new label-free method to determine the oxidation rate of peptides that is based on the consumption of the unoxidized peptide instead of measuring the formation of oxidized peptides. The reaction rate thus obtained presents a better linearity and lower variation when compared to the traditional method. The label-free method is also simpler to implement and automation can be achieved through label-free quantitation software.

Development of polymer-modified magnetic nanoparticles and quantum dots for Escherichia coli binding test

A rapid binding test has been developed for the detection of bacteria using polymer-modified magnetic nanoparticles. Polydopamine (PDA) can effectively act as a sorbent even in water solution, and a PDA coating on magnetic nanoparticles (MNPs) was therefore prepared to bind Escherichia coli (E. coli). Albeit non-selective, PDA-modified magnetic nanoparticles (MNPs@PDA) show nearly 100% efficiency in binding E. coli. If E. coli, grown in tryptic soy broth medium, is analyzed by capillary electrophoresis (CE) using phosphate buffer as the background electrolyte, two peaks are found, while a single peak is found with carbonate buffer containing 0.05% of poly(ethylene glycol). Self-polymerization of dopamine on E. coli at pH 9.5 is also feasible. The detection of E. coli is demonstrated by adding quantum dots (QDs) to form a QDs-PDA-E. coli aggregate for better CE analysis.

Ultrasensitive electrochemiluminescent detection of pentachlorophenol using a multiple amplification strategy based on a hybrid material made from quantum dots,graphene, and carbon nanotubes

We report on a highly sensitive and selective electrochemiluminescence (ECL) based method for the determination of pentachlorophenol (PCP). It is based on a new hybrid material composed of CdS quantum dots (QDs), graphene, and carbon nanotubes (CNTs), and uses peroxodisulfate as the coreactant. The use of this system results in a nearly 18-fold increase in ECL intensity. On interaction between PCP and the QDs, a decrease in ECL intensity is observed at PCP in a concentration as low as 1.0 pM and over a wide linear range (from 1.0 pM to 1.0 nM). The method is hardly affected by other chlorophenols and nitrophenols, and the electrode can be recycled.

Albumin coated CuInS2 quantum dots as a near-infrared fluorescent probe for NADH, and their application to an assay for pyruvate

Water-soluble CuInS₂ quantum dots (QDs) stabilized with 3-mercaptopropionic acid were synthesized in aqueous solution and then coated with bovine serum albumin. The resulting particles display fluorescence with a peak at 680 nm that is effectively quenched by 1, 4-dihydro-nicotinamide adenine dinucleotide (NADH), but not by 1, 4-nicotinamide adenine dinucleotide (NAD⁺). The enzyme lactate dehydrogenase catalyzes the reduction of pyruvate and dehydrogenation of lactic acid using NAD⁺ or NADH as a cosubstrate. The new QDs were applied to monitor the course of lactate dehydrogenase-catalyzed reaction of pyruvate by detecting NADH via its quenching effect. This resulted in a convenient and selective detection scheme for pyruvate. The detection limit is as low as 25 nM.

Specific biotinylation and sensitive enrichment of citrullinated peptides

Protein citrullination is a posttranslational modification where peptidylarginine is enzymatically deiminated to form peptidylcitrulline. Although the role of protein citrullination in both health and disease is being increasingly recognised, techniques available to identify citrullinated proteins and to map their citrullination site(s) are rare and often show poor sensitivity. Here, we present a sensitive technique for specific modification and selective enrichment of citrullinated peptides from complex biological samples. The technique is based on highly specific in-solution biotinylation of citrulline residues followed by selective enrichment of modified peptides using streptavidin beads. We demonstrate that a synthetic citrulline-containing peptide can be selectively enriched when less than 0.5 pmol is spiked into a highly heterogeneous peptide mixture. After enrichment, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of an aliquot of the streptavidin eluate corresponding to theoretically 50 fmol of the spiked-in peptide showed a prominent signal. We further demonstrate the sensitivity of our technique by enrichment of citrullinated peptides from enzymatically deiminated myelin basic protein (MBP), when 10 pmol was spiked into a heterogeneous biological digest. In MALDI-TOF MS analysis, six MBP-derived citrullinated peptides were observed, showing the efficiency of this enrichment strategy. The high sensitivity combined with the remarkable specificity of the described technique makes it a valuable tool for elucidating citrullination in various biological processes.

Radical Additions to Aromatic Residues in Peptides Facilitate Unexpected Side Chain and Backbone Losses

Accurate identification of fragments in tandem mass spectrometry experiments is aided by knowledge of relevant fragmentation mechanisms. Herein, novel radical addition reactions that direct unexpected side-chain dissociations at tryptophan and tyrosine residues are reported. Various mechanisms that can account for the observed dissociation channels are investigated by experiment and theory. The propensity for radical addition at a particular site is found to be primarily under kinetic control, which is largely dictated by molecular structure. In certain peptides, intramolecular radical addition reactions are favored, which leads to the observation of numerous unexpected fragments. In one pathway, radical addition leads to migration of an aromatic side chain to another residue. Alternatively, radical addition followed by hydrogen atom loss leads to cyclization of the peptide and increased observation of internal sequence fragments. Radical addition reactions should be considered when assigning fragmentation spectra obtained from activation of hydrogen deficient peptides.

Electrochemiluminescent sensing of dopamine using CdTe quantum dots capped with thioglycolic acid and supported with carbon nanotubes

We have synthesized water-dispersible CdTe quantum dots (QDs) capped with thioglycolic acid. Their quantum yield is higher than 54%. A sensitive electrochemiluminescence (ECL) method was established based on the modification of the composite of the QDs, carbon nanotubes and chitosan on indium tin oxide glass. The sensor displays efficient and stable anodic ECL which is quenched by dopamine. A respective sensor was designed that responds to dopamine linearly in the range of 50?pM to 10?nM, and the detection limit is 24?pM. Dopamine was determined with this sensor in spiked cerebro-spinal fluid with average recoveries of 95.7%.

Detection of silver(I) ion based on mixed surfactant-adsorbed CdS quantum dots

Mixed cationic and anionic surfactants were adsorbed on cadmium sulfide quantum dots (CdS QDs) capped with mercaptoacetic acid. The CdS QDs can be extracted into acetonitrile with 98 % efficiency in a single step. Phase separation only occurs at a molar ratio of 1:1.5 between cationic and anionic surfactants. The surfactant-adsorbed QDs in acetonitrile solution display stronger and more stable photoluminescence than in water solution. The method was applied for determination of silver(I) ion based on its luminescence enhancement of the QDs. Under the optimum conditions, the relative fluorescence intensity is linearly proportional to the concentration of silver(I) ion in the range between 50 pmol L^?1and 4 μmol L^?1, with a 20 pmol L^?1 detection limit. The relative standard deviation was 1.93 % for 9 replicate measurements of a 0.2 μmol L^?1 solution of Ag(I).