Conjugated Quantum Dots Research Articles

Iodinated PSMA Ligands as XFI Tracers for Targeted Cell Imaging and Characterization of Nanoparticles

Prostate cancer is the second most commonly diagnosed cancer in men worldwide. Despite this, current diagnostic tools are still not satisfactory, lacking sensitivity for early-stage or single-cell diagnosis. This study describes the development of small-molecule tracers for the well-known tumor marker prostate-specific membrane antigen (PSMA). These tracers contain a urea motif for PSMA-targeting and iodinated aromatic moieties to allow detection via X-ray fluorescence imaging (XFI). Tracers with a triiodobenzoyl moiety allowed the specific targeting and successful imaging of PSMA+ cell lines with XFI. The XFI-measured uptake of 7.88 × 10−18 mol iodine (I) per cell is consistent with the uptake of known PSMA tracers measured by other techniques such as inductively coupled plasma mass spectrometry (ICP-MS). This is the first successful application of XFI to tumor cell targeting with a small-molecule tracer. In addition, iodinated tracers were used for the characterization of quantum dots (QDs) conjugated to PSMA-targeting urea motifs. The resulting targeted QD conjugates were shown to selectively bind PSMA+ cell lines via confocal microscopy. The immobilized iodinated targeting vectors allowed the determination of the tracer/QD ratio via XFI and ICP-MS. This ratio is a key property of targeted particles and difficult to measure by other techniques.

Aptamer-Loop DNA Nanoflower Recognition and Multicolor Fluorescent Carbon Quantum Dots Labeling System for Multitarget Living Cell Imaging.

Visualization of multiple targets in living cells is important for understanding complex biological processes, but it still faces difficulties, such as complex operation, difficulty in multiplexing, and expensive equipment. Here, we developed a nanoplatform integrating a nucleic acid aptamer and DNA nanotechnology for living cell imaging. Aptamer-based recognition probes (RPs) were synthesized through rolling circle amplification, which were further self-assembled into DNA nanoflowers encapsulated by an aptamer loop. The signal probes (SPs) were obtained by conjugation of multicolor emission carbon quantum dots with oligonucleotides complementary to RPs. Through base pairing, RPs and SPs were hybridized to generate aptamer sgc8-, AS1411-, and Apt-based imaging systems. They were used for individual/simultaneous imaging of cellular membrane protein PTK7, nucleolin, and adenosine triphosphate (ATP) molecules. Fluorescence imaging and intensity analysis showed that the living cell imaging system can not only specifically recognize and efficiently bind their respective targets but also provide a 5-10-fold signal amplification. Cell-cycle-dependent distribution of nucleolin and concentration-dependent fluorescence intensity of ATP demonstrated the utility of the system for tracking changes in cellular status. Overall, this system shows the potential to be a simple, low-cost, highly selective, and sensitive living cell imaging platform.

Peptide Based Quantum Dots Conjugates as Promising Theranostic Candidates in Nanomedicines: Applications in Drug Delivery and Bioimaging

AbstractAdvancement in nanotechnology paves new way in nanomedicine via synthesis of various scaffolds bearing multifunctionalities having characteristics of both as diagnostic and therapeutic applications. Quantum dots (QDs) with exceptional luminescent properties exhibited some unbeatable characteristics of small size with ease in surface modifications and thus make them suitable candidates to be used in drug delivery and diagnosis of diseases. Meanwhile, various type of peptides are known in literature for their excellent therapeutic properties and thus make them promising candidates to be used as conjugate partner with the QDs. Highly luminescent properties and surface modifications flexibility of QDs, further urge to use them as promising conjugate partner with many small molecules including peptides and thus found applications in the field of nanomedicines. Thus Peptides ‐QDs conjugates exhibited the properties of both QDs and bioactivity, biocompatibility features of peptides and hence leave behind the limitation of both the individual partners (i. e. QDs and peptides). Thus the combined Peptides‐QDs conjugates acts as theranostic agent for various diseases where these acts as both imaging and drug delivery agent. Hence the present review focused on the theranostic applications of the Peptides‐QDs conjugates in various area of the nanomedicines. Also we focus on the area of improvement and advancement in nanomedicines for the diagnosis and treatment of the diseases in the early stages.

Fabrication of a low-cost benchtop optical imager for quantum dot microarray-based stress biomarker detection.

We report on a simplified optical imager to detect the presence of a stress biomarker protein, namely the Heat shock protein 90 (Hsp90). The imager consists of two elements the optical unit and the sensor, which is a custom-made biochip. Measurement is based on the masking of the streptavidin conjugated quantum dot's (Sav-QDs) fluorescence when Hsp90 attaches to it via biotinylated antibodies (Ab). The masking effect was directly proportional to the Hsp90 concentration. The cost-efficient benchtop imager developed comprises a CMOS sensor, standard optical lenses, and a narrow bandpass filter for optically eliminating background fluorescence. This approach is promising for the realization of cheap, robust, and reliable point-of-care detection systems for various biomarker analyses.

Quantum Dot-Fab' Conjugates as Compact Immunolabels for Microtubule Imaging and Cell Classification.

Antibodies and their conjugates of fluorescent labels are widely applied in life sciences research and clinical pathology. Among diverse label types, compact quantum dots (QDs) provide advantages of multispectral multiplexing, bright signals in the deep red and infrared, and low steric hindrance. However, QD-antibody conjugates have random orientation of the antigen-binding domain which may interfere with labeling and are large (20-30 nm) and heterogeneous, which limits penetration into biospecimens. Here, we develop conjugates of compact QDs and Fab' antibody fragments as primary immunolabels. Fab' fragments are conjugated site-specifically through sulfhydryl groups distal to antigen-binding domains, and the multivalent conjugates have small and homogeneous sizes (∼12 nm) near those of full-sized antibodies. Their performance as immunolabels for intracellular antigens is evaluated quantitatively by metrics of microtubule labeling density and connectivity in fixed cells and for cytological identification in fixed brain specimens, comparing results with probes based on spectrally-matched dyes. QD-Fab' conjugates outperformed QD conjugates of full-sized antibodies and could be imaged with bright signals with 1-photon and 2-photon excitation. The results demonstrate a requirement for smaller bioaffinity agents and site-specific orientation for the success of nanomaterial-based labels to enhance penetration in biospecimens and minimize nonspecific staining.

Deciphering breast cancer cell heterogeneity: a quantum dot-conjugate approach employing MCF-7 and THP-1 co-cultures

Cancer cell heterogeneity in MCF-7/THP-1 co-cultures was shown using a quantum dot antibody platform (QDλ/Ab), mimicking marker polarization in cancer cells during metastasis establishment.

A multiplex immunoprofiling approach for detecting the co-localization of breast cancer biomarkers using a combination of Alexafluor - Quantum dot conjugates and a panel of chromogenic dyes

There is a plethora of information embedded in a tissue section that the conventional IHC understands only partially. Predictive biomarkers for precision immuno-oncology heavily dependent on the spatial arrangement of cells and the co-expression patterns in the tissue sections. Here we have explored the versatility of indirect multiplex immunofluorescence (mIF) and indirect multiplex immunohistochemistry (mIHC) for the labeling of breast cancer prognostic markers in routinely processed, formalin-fixed paraffin-embedded (FFPE) tissues at high resolution. The multiplex immunohistochemistry protocol utilized sequential staining for the chromogenic immunolabelling of Estrogen Receptor α (ERα) or Progesterone Receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2), and Nucleoside diphosphate kinase 1 (NM23) by multicolor chromogens in different combinations. A feasible workflow for multiplex immunofluorescence was also effectively standardized for ERα, PR, and HER2 using combinations of commercially available Alexa Fluor and Quantum dots semiconductor nanocrystal conjugated secondary antibodies. Multiplex chromogenic immunolabeling revealed differential expression of the markers on the same slide. Kappa statistics revealed perfect agreement with uniplex immunohistochemistry. For multiplex fluorescence approach, surface receptor detection using Quantum dots and Alexa fluor dyes for cytoplasmic or nuclear markers performed well for profiling multiple co-localized biomarkers on a single paraffin tissue section. The technique developed reveals additional information such as co-expression, spatial relationships, and tumor heterogeneity, providing a deeper insight into developing combinatorial therapeutic strategies in clinical care. This high throughput workflow complements the outcomes of traditional IHC while saving tissue, time, labour, and reagents.

ICP-MS and fluorescence dual-mode detection of ZIKV-RNA based on quantum dot labeling with hybridization chain reaction

The detection of Zika virus (ZIKV) is of great significance to human life and health. Herein, we presented an ICP-MS and fluorescent dual-mode sensor for quantitative analysis of Zika virus RNA fragments (ZIKV-RNA), which employed quantum dots (QDs) as signal tags and combined with hybridization chain reaction (HCR). The dual-mode sensor realized cross-checking of the analysis results and improved the assay accuracy. Firstly, the single-stranded DNA (ssDNA) was anchored on the surface of magnetic beads (MBs). Afterward, HCR was conducted with probe DNA-CdSe quantum dots conjugates (pDNA-QDs) and link DNA (lDNA), producing the MBs-ssDNA-[pDNA-QDs-lDNA]n conjugates. In the presence of target ZIKV-RNA, a strand displacement reaction occurred, leading to the dissociation of the [pDNA-QDs-lDNA]n labels from the conjugates into the solution. Finally, the signal intensity was detected using ICP-MS and fluorescence analysis, with achieved limits of detection of 131 pM and 152 pM, respectively. The inter-assay RSD values of fluorescence and ICP-MS were 3.94 % and 4.26 % at 10 nM level, respectively, showing that the method had good precision. This method showed high selectivity and was applied to the analysis of biological fluids. There was no significant difference between the results of ICP-MS modes and fluorescence mode. This method offers a new strategy for sensitivity analysis of ZIKV-RNA and exhibits promise in clinical applications for diagnosis.

A fluorescent quantum dot conjugate to probe the interaction of Enterolobium contortisiliquum trypsin inhibitor with cancer cells

Serine proteases play crucial biological roles and have their activity controlled by inhibitors, such as the EcTI, a serine protease inhibitor purified from Enterolobium contortisiliquum seeds, which has anticancer activity. This study aimed to conjugate EcTI with quantum dots (QDs), fluorophores with outstanding optical properties, and investigate the interaction of QDs-EcTI nanoprobe with cancer cells. The conjugation was evaluated by fluorescence correlation spectroscopy (FCS) and fluorescence microplate assay (FMA). EcTI inhibitory activity after interaction with QDs was also analyzed. From FCS, the conjugate presented a hydrodynamic diameter about 4× greater than bare QDs, suggesting a successful conjugation. This was supported by FMA, which showed a relative fluorescence intensity of ca. 3815% for the nanosystem, concerning bare QDs or EcTI alone. The EcTI inhibitory activity remained intact after its interaction with QDs. From flow cytometry analyses, approximately 62% of MDA-MB-231 and 90% of HeLa cells were labeled with the QD-EcTI conjugate, suggesting that their membranes have different protease levels to which EcTI exhibits an affinity. Concluding, the QD-EcTI represents a valuable nanotool to study the interaction of this inhibitor with cancer cells using fluorescence-based techniques with the potential to unravel the intricate dynamics of interplays between proteases and inhibitors in cancer biology.

Novel Förster Resonance Energy Transfer probe with quantum dot for a long-time imaging of active caspases inside individual cells

With the goal to investigate biological phenomena at a single-cell level, we designed, synthesized and tested a molecular probe based on Förster resonance energy transfer (FRET) between a highly luminescent quantum dot (QD) as a donor and a fluorophore or fluorescence quencher as an acceptor linked by a specific peptide. In principle, QD luminescence, effectively dissipated in the probe, is switched on after the cleavage of the peptide by a protease and the release of the quencher. We proposed a novel synthesis strategy of a probe. A two-step synthesis consists of: (i) Conjugation of CdTe QDs functionalized by –COOH groups of succinic acid on the nanoparticle surface with the designed specific peptide (GTADVEDTSC) using a ligand-exchange approach; (ii) A fast, high-yield reaction of amine-reactive succinimidyl group on the BHQ-2 quencher with N-terminal of the peptide. This way, any crosslinking between individual nanoparticles and any nonspecific conjugation bonds are excluded. The analysis of the product after the first step proved a high reaction yield and nearly no occurrence of unreacted QDs, a prerequisite of the specificity of our luminescent probe. Its parameters evaluated as Michaelis-Menten description of enzymatic kinetics are similar to products published by other groups. Our research is focused on the fluorescence microscopy analyses of biologically active molecules, such as proteolytic active caspases, playing important roles in cell signaling regulations in normal and diseased states. Consequently, they are attractive targets for clinical diagnosis and medical therapy. The ultimate goal of our work was to synthesize a new QD luminescent probe for a long-time quantitative monitoring of active caspase-3/7 distribution in apoptotic osteoblastic MC3T3-E1 cells treated with camptothecin. As a result of comparison, our synthetized luminescent probe provides longer imaging times of caspases than commercial products. The probe proved the stability of the luminescence signal inside cells for more than 14 days.

Nanoprobes based on fluorescent semiconductor nanocrystals and single-domain antibodies for highly sensitive detection of epidermal growth factor receptor in tumor cells

Background. The development of highly oriented conjugates of quantum dots (QDs) and single-domain antibodies (sdAbs) as innovative fluorescence imaging nanoprobes that specifically recognize tumor biomarkers, in particular, epidermal growth factor receptor (EGFR), is a promising approach to improving immunohistochemical tumor typing.Aim. The study was aimed at developing fluorescent nanoprobes based on QDs and sdAbs that specifically recognize EGFR, as well as evaluating their functional characteristics (size and optical properties) and functional activity.Materials and methods. QDs were obtained using high-temperature organometallic synthesis and transferred into the aqueous phase by means of stepwise replacement of ligands on the QD surface. The QDs and sdAbs were conjugated in an oriented manner using a bifunctional cross-linking agent. Detailed characteristics of the resulting conjugates were analyzed by the dynamic light scattering and immunoassay methods. Functional activity was assessed on the model human epidermoid carcinoma cells line A431.Results. The QD–sdAb conjugates have been standardized in terms of control parameters determining their functional activity, in particular, hydrodynamic diameter and efficiency of binding with target tumor cells. They are characterized by high dispersity, homogeneity, and specific functional activity towards their molecular target.Conclusion. The results demonstrate the potential use of the designed QD–sdAb conjugates for EGRF detection in immunohistochemical typing of tumor.

In vivo imaging of prostate tumor-targeted folic acid conjugated quantum dots

Cancer is a major threat to human health; thus, early detection is imperative for successful management. Rapid diagnosis can be achieved by imaging primary (subcutaneous) tumors using fluorophores conjugated with tumor markers. Here, the application of biocompatible, quantum efficient, monodisperse, and photostable polymer-coated quantum dots (PQDs) is demonstrated for targeted prostate tumor imaging in living SCID mice. Briefly, PQDs (blue) are conjugated to folic acid (FA-PQDs) using DCC-NHS chemistry. Initially, in vitro targeted imaging via FA-PQDs is evaluated in LNCaP cells. The confocal microscopic evaluation demonstrates the uptake of FA-PQDs. To understand the dispersion of PQDs in vivo, the biodistribution of PQDs is assessed at different time intervals (1- 180 min) using whole-body fluorescence imaging and computed tomography (CT) scan. PQDs are seen to accumulate in organs like the liver, kidneys, spleen, lungs, and urinary bladder within 60 min, however, PQDs are not observed at 180 min indicating renal clearance. Further, to target the prostate tumor (~ 200 mm3) in mice, FA-PQDs are injected intravenously, and whole-body fluorescence imaging along with a CT scan is recorded. FA-PQDs are seen at the tumor site as compared to PQDs. The results confirm that the FA-PQDs function as excellent nanoprobes for targeted tumor imaging in vivo.

Fluorescence Masking Based Multifunctional Quantum Dots’ Assay for HSP90α Interactions Detection

HSP90α is one of the most common stress proteins in cells; hence, it is a good target for developing drugs and testing systems for cancer or physical stress levels in humans. Streptavidin conjugated quantum dots (Sav-QDs) are widely used as fluorophores for biosensing to overcome chemical labelling problems. In this work, we have attempted to develop a multifunctional and robust assay for HSP90α. The detection technique was based on the masking of the fluorescence of spotted Sav-QDs on nitrocellulose chips (NC). Biotinylated ligand/antibody attaches to the spotted Sav-QD and then HSP90α is attached, which causes the masking of fluorescence. The masking of fluorescence was used to detect protein–ligand interactions, the effect of inhibitors, protein–protein interactions, and the presence of protein in the biological sample. The load of detection (LoD) of the assay lies in the nano molar range, making it a sensitive assay. The results from the experiments suggest that the used approach is promising for developing a multifunctional, robust, and sensitive assay for proteins that can be used for point-of-care detection in complex biological samples.

MicroLED biosensor with colloidal quantum dots and smartphone detection.

A fluorescence sensor with the capability for spatially multiplexed measurements utilizing smartphone detection is presented. Bioconjugated quantum dots are used as the fluorescent tag and are excited using a blue-emitting microLED (µLED). The 1-dimensional GaN µLED array is butt-coupled to one edge of the glass slide to take advantage of total internal reflection fluorescence (TIRF) principles. The bioassays on the top surface of the glass waveguide are excited and the resultant fluorescence is detected with the smartphone. The red, green, and blue channels of the digital image are utilized to spectrally separate the excitation light from the fluorescence for analysis. Using a biotin-functionalized glass slide as proof of principle, we have shown that streptavidin conjugated quantum dots can be detected down to a concentration of 8 nM.

Quantum Dot–Organic Molecule Conjugates as Hosts for Photogenerated Spin Qubit Pairs

The inherent spin polarization present in photogenerated spin-correlated radical pairs makes them promising candidates for quantum computing and quantum sensing applications. The spin states of these systems can be probed and manipulated with microwave pulses using electron paramagnetic resonance spectrometers. However, to date, there are no reports on magnetic resonance-based spin measurements of photogenerated spin-correlated radical pairs hosted on quantum dots. In the current work, we prepare dye molecule-inorganic quantum dot conjugates and show that they can produce photogenerated spin-polarized states. The dye molecule, D131, is chosen for its ability to undergo efficient charge separation, and the nanoparticle materials, ZnO quantum dots, are chosen for their promising spin properties. Transient and steady state optical spectroscopy performed on ZnO quantum dot-D131 conjugates shows that reversible photogenerated charge separation is occurring. Transient and pulsed electron paramagnetic resonance experiments are then performed on the photogenerated radical pair, which demonstrate that (1) the radical pair is polarized at moderate temperatures and well modeled by existing theories and (2) the spin states can be accessed and manipulated with microwave pulses. This work opens the door to a new class of promising qubit materials that can be photogenerated in polarized states and hosted by highly tailorable inorganic nanoparticles.

Quantum Dot-Peptide Conjugates as Energy Transfer Probes for Sensing the Proteolytic Activity of Matrix Metalloproteinase-14.

We detail the assembly and characterization of quantum dot (QD)-dye conjugates constructed using a peptide bridge specifically designed to recognize and interact with a breast cancer biomarker─matrix metalloproteinase-14 (MMP-14). The assembled QD conjugates are then used as optically addressable probes, relying on Förster resonance energy transfer (FRET) interactions as a transduction mechanism to detect the activity of MMP-14 in solution phase. The QDs were first coated with dithiolane poly(ethylene glycol) (PEG) bearing a carboxyl group that allows coupling via amide bond formation with different dye-labeled peptides. The analytical capability of the conjugates is enabled by correlating changes in the FRET efficiency with the conjugate valence and/or QD-to-dye separation distance, triggered and modulated by enzymatic proteolysis of surface-tethered peptides. The FRET probe exhibits great sensitivity to enzyme digestion with sub-nanomolar limit of detection. We further analyze the proteolysis data within the framework of the Michaelis-Menten model, which considers the fact that surface-attached peptides have a slower diffusion coefficient than free peptides. This results in reduced collision frequency and lower catalytic efficiency, kcat/KM. Our results suggest that our conjugate design is promising, effective, and potentially useful for in vivo analysis.

Conjugation of Quantum Dot (CdS) with Antibodies for Identification of Ornithobacterium Rhinotracheale

Conjugation of Quantum Dot (CdS) with Antibodies for Identification of Ornithobacterium Rhinotracheale

New Quantum-Dot-Based Fluorescent Immunosensor for Cancer Biomarker Detection

Cancer antigen 15-3 (CA 15-3) is a biomarker for breast cancer used to monitor response to treatments and disease recurrence. The present work demonstrates the preparation and application of a fluorescent biosensor for ultrasensitive detection of the cancer antigen CA 15-3 protein tumor marker using mercaptopropionic-acid-functionalized cadmium telluride (CdTe@MPA) quantum dots (QDs) conjugated with CA 15-3 antibodies. First, the QDs were synthesized by the hydrothermal route, resulting in spherical nanoparticles up to 3.50 nm in diameter. Subsequently, the QD conjugates were characterized by Fourier transform infrared spectroscopy (FTIR), UV absorption, and fluorescence. The interaction between the conjugates and the protein was studied by fluorescence spectroscopy in buffer and in 10-fold diluted commercial human serum. Calibration in spiked serum samples gave a detection limit of 0.027 U/mL, 1000-fold lower than the clinical limit for CA 15-3 (25 U/mL to 30 U/mL), indicating that this is an ultrasensitive technique. In addition, a rapid response was obtained within 10 min. The biosensor was selective in the presence of the interfering serum proteins BSA, CEA, and CA-125, with a maximum interference of 2% for BSA. The percent recovery was close to 100% with maximum relative standard deviation (RSD%) values of 1.56. Overall, the developed CA 15-3 biosensor provides a simple and sensitive method for ultrasensitive monitoring of breast cancer, as well as the ability to detect other molecules of interest in human serum matrices.

Dual-targeting magnetic fluorescent mesoporous organosilicon hollow nanospheres for gambogic acid loading, sustained release and anti-tumor properties

Hydrophilic mesoporous organosilica nanoparticles (HMNPs) were synthesized by introducing Fe3O4 on the basis of mesoporous organosilicon nanoparticle hollow spheres as a drug carrier. N- and S- doped graphene quantum dots (NS-GQDs) and polyethylenimine (PEI) were grafted onto the surface of HMNPs. Finally, two types of composite mesoporous particles, HMNPs-NS-GQDs-PEI-HA and HMNPs-NS-GQDs-PEI-FA which can enter VX2 cells for fluorescence confocal imaging, were further modified by targeting ligand hyaluronic acid (HA) and folic acid (FA). The heteroatoms of NS-GQDs have intelligent functional effects, which can not only improve the photoluminescence performance of GQDs, but also generate conjugated quantum dots with GA, so as to synergistically improve the drug effectiveness. Under illumination by near-infrared (NIR) radiation, gambogic acid (GA)-loaded HMNPs was promoted to the sustained release of gambogic acid. The loading and sustained release of the anticancer drug GA on the compound mesoporous particles were studied. The cytotoxicity and antitumor properties of the compound mesoporous particles were investigated by both in vitro cell experiments and in vivo experiments in mice. The results showed that HMNPs-NS-GQDs-PEI-HA and HMNPs-NS-GQDs-PEI-FA have desired fluorescence and magnetic properties for cell fluorescence imaging and magnetic targeting of cancer cells. Both of composite mesoporous particles have desired loading capacity on GA and pH-responsive drug release behavior as well as low cytotoxicity and good biocompatibility. The cell survival rate of HMNPs-NS-GQDs-PEI-HA and HMNPs-NS-GQDs-PEI-FA loaded with GA was only 7.69% and 8.71%, respectively, after 24 h, which can effectively induce the apoptosis of VX2 cells. Tumor experiments in mice demonstrated that HMNPs-NS-GQDs-PEI-HA inhibits tumor growth and exhibits magnetic targeting function.

Quantum Dot-Antibody Conjugates for Immunofluorescence Studies of Biomolecules and Subcellular Structures.

Quantum dots, or nanoscale semiconductors, are one of the most important materials for various research and development purposes. Due to their advantageous photoluminescence and electronic properties, namely, their unique photostability, high brightness, narrow emission spectra from visible to near-infrared wavelengths, convey them significant advantages over widely used fluorochromes, including organic dyes, fluorescent probes. Quantum dots are a unique instrument for a wide range of immunoassays with antibodies. The paper provides an overview of the developed and already applied methods of quantum dot surface modification, quantum dots conjugation to different antibodies (non-covalent, direct covalent linkage or with the use of special adapter molecules), as well as practical examples of recent quantum dot-antibody applications in the immunofluorescence microscopy for cell and cell structure imaging, fluorescent assays for biomolecules detection and in diagnostics of various diseases. The review presents advantages of quantum dot-antibody conjugation technology over the existing methods of immunofluorescence studies and a forward look into its potential prospects in biological and biomedical research.