ABSTRACT
Understanding the mechanisms of assembly and disassembly of macromolecular structures in cells relies on solving biomolecular interactions. However, those interactions often remain unclear because tools to track molecular dynamics are not sufficiently resolved in time or space. In this study, we present a straightforward method for resolving inter- and intra-molecular interactions in cell adhesive machinery, using quantum dot (QD) based Förster resonance energy transfer (FRET) nanosensors. Using a mechanosensitive protein, talin, one of the major components of focal adhesions, we are investigating the mechanosensing ability of proteins to sense and respond to mechanical stimuli. First, we quantified the distances separating talin and a giant unilamellar vesicle membrane for three talin variants. These variants differ in molecular length. Second, we investigated the mechanosensing capabilities of talin, i.e., its conformational changes due to mechanical stretching initiated by cytoskeleton contraction. Our results suggest that in early focal adhesion, talin undergoes stretching, corresponding to a decrease in the talin-membrane distance of 2.5 nm. We demonstrate that QD-FRET nanosensors can be applied for the sensitive quantification of mechanosensing with a sub-nanometer accuracy.
ABSTRACT
Integrating isothermal nucleic acid amplification strategies into immunoassays can significantly decrease analytical limits of detection (LODs). On the other hand, an amplification step adds time, complication, reagents, and costs to the assay format. To evaluate the pros and cons in the context of heterogeneous multistep immunoassays, we quantified prostate-specific antigen (PSA) with and without rolling circle amplification (RCA). In addition, we compared time-gated (TG) with continuous-wave (CW) photoluminescence (PL) detection using a terbium complex and a fluorescein dye, respectively. For both direct (non-amplified) and amplified assays, TG PL detection provided circa four- to eightfold lower LODs, illustrating the importance of autofluorescence background suppression even for multi-wash assay formats. Amplified assays required an approximately 2.4 h longer assay time but led to almost 100-fold lower LODs down to 1.3 pg/mL of PSA. Implementation of TG-FRET (using a Tb-Cy5.5 donor-acceptor pair) into the RCA immunoassay resulted in a slightly higher LOD (3.0 pg/mL), but the ratiometric detection format provided important benefits, such as higher reproducibility, lower standard deviations, and multiplexing capability. Overall, our direct comparison demonstrated the importance of biological background suppression even in heterogeneous assays and the potential of using isothermal RCA for strongly decreasing analytical LODs, making such assays viable alternatives to conventional enzyme-linked immunosorbent assays (ELISAs).
ABSTRACT
Human cystatin C (hCC) is a physiologically important protein that serves as intra- and extracellular cysteine proteinase inhibitor in homeostasis. However, in pathological states it dimerizes and further oligomerizes accumulating into a toxic amyloid. HCC forms an active monomer in the extracellular space and becomes an inactive dimer when internalized in cellular organelles. However, hCC cell penetration and its oligomeric state during this process are not well understood. To determine if and how the oligomeric state influences hCC transmembrane migration, we investigated the internalization of the hCC wild type protein as well as three different mutants, which exclusively exist in the monomeric or multimeric state into HeLa cells via confocal fluorescence microscopy. Our results showed that the preferred pathway was endocytosis and that the oligomeric state did not significantly influence the internalization because both monomeric and dimeric hCC migrated into HeLa cells. Considering the differences of the active monomeric and the passive dimeric states of hCC, our findings contribute to a better understanding of the intra and extra cellular functions of hCC and their interaction with cysteine proteases.
ABSTRACT
Almost all pathogens, whether viral or bacterial, utilize key proteolytic steps in their pathogenesis. The ability to detect a pathogen's genomic material along with its proteolytic activity represents one approach to identifying the pathogen and providing initial evidence of its viability. Here, we report on a prototype biosensor design assembled around a single semiconductor quantum dot (QD) scaffold that is capable of detecting both nucleic acid sequences and proteolytic activity by using orthogonal energy transfer (ET) processes. The sensor consists of a central QD assembled via peptidyl-PNA linkers with multiple DNA sequences that encode complements to genomic sequences originating from the Ebola, Influenza, and COVID-19 viruses, which we use as surrogate targets. These are hybridized to complement strands labeled with a terbium (Tb) chelate, AlexaFluor647 (AF647), and Cy5.5 dyes, giving rise to two potential FRET cascades: the first includes Tb â QD â AF647 â Cy5.5 (â = ET step), which is detected in a time-gated modality, and QD â AF647 â Cy5.5, which is detected from direct excitation. The labeled DNA-displaying QD construct is then further assembled with a RuII-modified peptide, which quenches QD photoluminescence by charge transfer and is recognized by a protease to yield the full biosensor. Each of the labeled DNAs and peptides can be ratiometrically assembled to the QD in a controllable manner to tune each of the ET pathways. Addition of a given target DNA displaces its labeled complement on the QD, disrupting that FRET channel, while protease addition disrupts charge transfer quenching of the central QD scaffold and boosts its photoluminescence and FRET relay capabilities. Along with characterizing the ET pathways and verifying biosensing in both individual and multiplexed formats, we also demonstrate the ability of this construct to function in molecular logic and perform Boolean operations; this highlights the construct's ability to discriminate and transduce signals between different inputs or pathogens. The potential application space for such a sensor device is discussed.
Subject(s)
Biosensing Techniques , Carbocyanines , Quantum Dots , Quantum Dots/chemistry , Peptide Hydrolases/metabolism , Fluorescence Resonance Energy Transfer , Peptides/chemistry , DNA/chemistry , Endopeptidases/metabolismABSTRACT
DNA nanotechnology has now enabled the self-assembly of almost any prescribed 3-dimensional nanoscale structure in large numbers and with high fidelity. These structures are also amenable to site-specific modification with a variety of small molecules ranging from drugs to reporter dyes. Beyond obvious application in biotechnology, such DNA structures are being pursued as programmable nanoscale optical breadboards where multiple different/identical fluorophores can be positioned with sub-nanometer resolution in a manner designed to allow them to engage in multistep excitonic energy-transfer (ET) via Förster resonance energy transfer (FRET) or other related processes. Not only is the ability to create such complex optical structures unique, more importantly, the ability to rapidly redesign and prototype almost all structural and optical analogues in a massively parallel format allows for deep insight into the underlying photophysical processes. Dynamic DNA structures further provide the unparalleled capability to reconfigure a DNA scaffold on the fly in situ and thus switch between ET pathways within a given assembly, actively change its properties, and even repeatedly toggle between two states such as on/off. Here, we review progress in developing these composite materials for potential applications that include artificial light harvesting, smart sensors, nanoactuators, optical barcoding, bioprobes, cryptography, computing, charge conversion, and theranostics to even new forms of optical data storage. Along with an introduction into the DNA scaffolding itself, the diverse fluorophores utilized in these structures, their incorporation chemistry, and the photophysical processes they are designed to exploit, we highlight the evolution of DNA architectures implemented in the pursuit of increased transfer efficiency and the key lessons about ET learned from each iteration. We also focus on recent and growing efforts to exploit DNA as a scaffold for assembling molecular dye aggregates that host delocalized excitons as a test bed for creating excitonic circuits and accessing other quantum-like optical phenomena. We conclude with an outlook on what is still required to transition these materials from a research pursuit to application specific prototypes and beyond.
Subject(s)
Fluorescence Resonance Energy Transfer , Quantum Dots , Quantum Dots/chemistry , Biotechnology , Fluorescent Dyes/chemistry , DNA/chemistryABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the global coronavirus disease 2019 (COVID-19) pandemic, has caused well over 750 million infections and 6.8 million deaths. Rapid diagnosis and isolation of infected patients are the primary aims of the concerned authorities to minimize the casualties. The endeavor to mitigate the pandemic has been impeded by the emergence of newly identified genomic variants of SARS-CoV-2. Some of these variants are considered as serious threats because of their higher transmissibility and potential immune evasion, leading to reduced vaccine efficiency. Nanotechnology can play an important role in advancing both diagnosis and therapy of COVID-19. In this review, nanotechnology-based diagnostic and therapeutic strategies against SARS-CoV-2 and its variants are introduced. The biological features and functions of the virus, the mechanism of infection, and currently used approaches for diagnosis, vaccination, and therapy are discussed. Then, nanomaterial-based nucleic acid- and antigen-targeting diagnostic methods and viral activity suppression approaches that have a strong potential to advance both diagnostics and therapeutics toward control and containment of the COVID-19 pandemic are focused upon.
Subject(s)
COVID-19 , Nanostructures , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , COVID-19/therapy , Pandemics/prevention & control , Nanotechnology , COVID-19 TestingABSTRACT
Upconversion nanoparticles (UCNPs) are some of the most promising nanomaterials for bioanalytical and biomedical applications. One important challenge to be still solved is how UCNPs can be optimally implemented into Förster resonance energy transfer (FRET) biosensing and bioimaging for highly sensitive, wash-free, multiplexed, accurate, and precise quantitative analysis of biomolecules and biomolecular interactions. The many possible UCNP architectures composed of a core and multiple shells doped with different lanthanoid ions at different ratios, the interaction with FRET acceptors at different possible distances and orientations via biomolecular interaction, and the many and long-lasting energy transfer pathways from the initial UCNP excitation to the final FRET process and acceptor emission make the experimental determination of the ideal UCNP-FRET configuration for optimal analytical performance a real challenge. To overcome this issue, we have developed a fully analytical model that requires only a few experimental configurations to determine the ideal UCNP-FRET system within a few minutes. We verified our model via experiments using nine different Nd-, Yb-, and Er-doped core-shell-shell UCNP architectures within a prototypical DNA hybridization assay using Cy3.5 as an acceptor dye. Using the selected experimental input, the model determined the optimal UCNP out of all theoretically possible combinatorial configurations. An extreme economy of time, effort, and material was accompanied by a significant sensitivity increase, which demonstrated the powerful feat of combining a few selected experiments with sophisticated but rapid modeling to accomplish an ideal FRET biosensor.
Subject(s)
Lanthanoid Series Elements , Nanoparticles , Nanostructures , Fluorescence Resonance Energy Transfer/methodsABSTRACT
Plasmonic metal nanoparticles and semiconductor quantum dots (QDs) are two of the most widely applied nanomaterials for optical biosensing and bioimaging. While their combination for fluorescence quenching via nanosurface energy transfer (NSET) or Förster resonance energy transfer (FRET) offers powerful ways of tuning and amplifying optical signals and is relatively common, metal-QD nanohybrids for plasmon-enhanced fluorescence (PEF) have been much less prevalent. A major reason is the competition between fluorescence quenching and enhancement, which poses important challenges for optimizing distances, orientations, and spectral overlap toward maximum PEF. In this feature article, we discuss the interplay of the different quenching and enhancement mechanisms (a mixed distance dependence of quenching and enhancement - "quenchancement") to better understand the obstacles that must be overcome for the development of metal-QD nanohybrid-based PEF biosensors. The different nanomaterials, their combination within various surface and solution based design concepts, and their structural and photophysical characterization are reviewed and applications toward advanced optical biosensing and bioimaging are presented along with guidelines and future perspectives for sensitive, selective, and versatile bioanalytical research and biomolecular diagnostics with metal-QD nanohybrids.
Subject(s)
Biosensing Techniques , Quantum Dots , Semiconductors , Fluorescence Resonance Energy Transfer/methods , Metals , Biosensing Techniques/methodsABSTRACT
Upconversion nanoparticles (UCNPs) have been frequently applied in Förster resonance energy transfer (FRET) bioanalysis. However, the understanding of how surface coatings, bioconjugation, and dye-surface distance influence FRET biosensing performance has not significantly advanced. Here, we investigated UCNP-to-dye FRET DNA-hybridization assays in H2O and D2O using â¼24 nm large NaYF4:Yb3+,Er3+ UCNPs coated with thin layers of silica (SiO2) or poly(acrylic acid) (PAA). FRET resulted in strong distance-dependent PL intensity changes. However, the PL decay times were not significantly altered because of continuous Yb3+-to-Er3+ energy migration during Er3+-to-dye FRET. Direct bioconjugation of DNA to the thin PAA coating combined with the closest possible dye-surface distance resulted in optimal FRET performance with minor influence from competitive quenching by H2O. The better comprehension of UCNP-to-dye FRET was successfully translated into a microRNA (miR-20a) FRET assay with a limit of detection of 100 fmol in a 80 µL sample volume.
Subject(s)
Biosensing Techniques , Nanoparticles , Nucleic Acids , Fluorescence Resonance Energy Transfer , Silicon Dioxide , Biosensing Techniques/methodsABSTRACT
Lanthanide(III) (Ln3+) complexes feature desirable luminescence properties for cell microscopy imaging, but cytosolic delivery of Ln3+ complexes and their use for 2P imaging of live cells are challenging. In this article, we describe the synthesis and spectroscopic characterizations of a series of Ln3+ complexes based on two ligands, L1 and L2, featuring extended picolinate push-pull antennas for longer wavelength absorption and 2P absorption properties as well as a free carboxylate function for conjugation to peptides. Several cell penetrating peptide/Ln3+ complex conjugates were then prepared with the most interesting luminescent complexes, Tb(L1) and Eu(L2), and with two cell penetrating peptides (CPPs), ZF5.3 and TP2. A spectroscopic analysis demonstrates that the luminescence properties of the complexes are not affected by conjugation to the peptide. The conjugates were evaluated for one-photon (1P) time-gated microscopy imaging, which suppresses biological background fluorescence, and 2P confocal microscopy. Whereas TP2-based conjugates were unable to enter cells, successful 1P and 2P imaging was performed with ZF5.3[Tb(L1)]. 2P confocal imaging suggests proper internalization and cytosolic delivery as expected for this CPP. Noteworthy, 2P confocal microscopy also allowed characterization of the luminescence properties of the complex (spectrum, lifetime) within the cell, opening the way to functional luminescent probes for 2P confocal imaging of live cells.
Subject(s)
Lanthanoid Series Elements , Lanthanoid Series Elements/chemistry , Luminescence , Microscopy/methods , Photons , Ligands , PeptidesABSTRACT
Long-lived emissive nucleic acid probes are widely used in biochemical analysis due to their programmable structures, high signal-to-background ratio, and high sensitivity. Homogeneous detection based on long-lived emissive nucleic acid probes is often achieved through Förster resonance energy transfer (FRET), which suffers from the limitation of a narrow effective distance range. Herein, a new strategy of accessing nucleic acid hybridization-responsive luminescent probes is presented. The photoluminescence (PL) of a Lumi4-Tb complex internally modified with DNA is switched on by nucleic acid hybridization, after which the PL is increased up to 20 times. PL lifetime analysis revealed a possible mechanism of luminescence enhancement. Due to the flexibility of single-stranded nucleic acid chains, the bases and phosphate groups can coordinate with the Tb(III), which reduces the stability of the Tb complex and results in weak PL. After hybridization, the rigid double helix structure suppresses the coordination between Tb(III) and the bases or phosphate groups, causing luminescence enhancement. As the DNA sequence can be freely designed, an array of probes for different DNA or RNA targets can be created with the same Tb complex. Moreover, the novel probe design can afford pM detection limits of DNA or RNA without any nucleic acid amplification and exhibits great potential for nucleic acid detection in clinical diagnosis.
Subject(s)
Luminescence , Nucleic Acids , RNA , Nucleic Acid Hybridization/methods , DNA/chemistry , Nucleic Acid Probes , PhosphatesABSTRACT
Auranofin (Ridaura®, AUF) is a gold complex originally approved as an antirheumatic agent that has emerged as a potential candidate for multiple repurposed therapies. The best-studied anticancer mechanism of AUF is the inhibition of thioredoxin reductase (TrxR). However, a number of reports indicate a more complex and multifaceted mode of action for AUF that could be cancer cell type- and dose-dependent. In this study, we observed that AUF displayed variable cytotoxicity in five triple-negative breast cancer cell lines. Using representative MDA-MB-231 cells treated with moderate and cytotoxic doses of AUF, we evidenced that an AUF-mediated TrxR inhibition alone may not be sufficient to induce cell death. Cytotoxic doses of AUF elicited rapid and drastic intracellular oxidative stress affecting the mitochondria, cytoplasm and nucleus. A "redoxome" proteomics investigation revealed that a short treatment with a cytotoxic dose AUF altered the redox state of a number of cysteines-containing proteins, pointing out that the cell proliferation/cell division/cell cycle and cell-cell adhesion/cytoskeleton structure were the mostly affected pathways. Experimentally, AUF treatment triggered a dose-dependent S-phase arrest and a rapid disintegration of the actin cytoskeleton structure. Our study shows a new spectrum of AUF-induced early effects and should provide novel insights into the complex redox-based mechanisms of this promising anticancer molecule.
ABSTRACT
Biosensing approaches that combine small, engineered antibodies (nanobodies) with nanoparticles are often complicated. Here, we show that nanobodies with different C-terminal tags can be efficiently attached to a range of the most widely used biocompatible semiconductor quantum dots (QDs). Direct implementation into simplified assay formats was demonstrated by designing a rapid and wash-free mix-and-measure immunoassay for the epidermal growth factor receptor (EGFR). Terbium complex (Tb)-labeled hexahistidine-tagged nanobodies were specifically displaced from QD surfaces via EGFR-nanobody binding, leading to an EGFR concentration-dependent decrease of the Tb-to-QD Förster resonance energy transfer (FRET) signal. The detection limit of 80±20â pM (16±4â ng mL-1 ) was 3-fold lower than the clinical cut-off concentration for soluble EGFR and up to 10-fold lower compared to conventional sandwich FRET assays that required a pair of different nanobodies.
Subject(s)
Quantum Dots , Single-Domain Antibodies , ErbB Receptors , Fluorescence Resonance Energy Transfer , TerbiumABSTRACT
Histamine (HA) is an indicator of food freshness and quality. However, high concentrations of HA can cause food poisoning. Simple, rapid, sensitive, and specific quantification can enable efficient screening of HA in food and beverages. However, conventional assays are complicated and time-consuming, as they require multiple incubation, washing, and separation steps. Here, we demonstrate that time-gated Förster resonance energy transfer (TG-FRET) between terbium (Tb) complexes and organic dyes can be implemented in both immunosensors and aptasensors for simple HA quantification using a rapid, single-step, mix-and-measure assay format. Both biosensors could quantify HA at concentrations relevant in food poisoning with limits of detection of 0.19 µg/mL and 0.03 µg/mL, respectively. Excellent specificity was documented against the structurally similar food components tryptamine and l-histidine. Direct applicability of the TG-FRET assays was demonstrated by quantifying HA in spiked fish and wine samples with both excellent concentration recovery and agreement with conventional multistep enzyme-linked immunosorbent assays (ELISAs). Our results show that the simplicity and rapidity of TG-FRET assays do not compromise sensitivity, specificity, and reliability, and both immunosensors and aptasensors have a strong potential for their implementation in advanced food safety screening.
Subject(s)
Biosensing Techniques , Foodborne Diseases , Animals , Antibodies , Fluorescence Resonance Energy Transfer/methods , Histamine , Immunoassay , Oligonucleotides , Reproducibility of ResultsABSTRACT
Luminescent upconversion nanocrystals (UCNCs) have become one of the most promising nanomaterials for biosensing, imaging, and theranostics. However, their ultimate translation into robust luminescent probes for daily use in biological and medical laboratories requires comprehension and control of the many possible deactivation pathways that cause upconversion luminescence (UCL) quenching. Here, we demonstrate that thorough modeling of UCL rise and decay kinetics using a freely accessible software can identify the UCL quenching mechanisms in small (<40 nm) UCNCs with spatial and temporal resolution. Applied to the most relevant ß-NaYF4:Yb3+,Er3+ UCNCs, our model showed that only a few distinct nonradiative low-energy transitions were deactivated via specific solvent and ligand vibrations with a strong downstream effect on the population and depopulation dynamics of the emitting states. UCL quenching could penetrate ca. 4 nm inside the UCNC, which resulted in significant size-dependent changes of UCL intensities and spectra. Despite the large surface-to-volume ratios and UCL quenching via the UCNC surface, we found strong contributions of the outer layers to the overall UCL, which will be highly important for the design of UCNPs to investigate biomolecular interactions via distance-dependent energy transfer methods. Our advanced kinetic model is easily scalable to different UCNC architectures, environments, and energy transfer interactions such that relatively simple modeling of UCL kinetics can be used for efficiently optimizing UCNCs for their final application as practical luminescent probes.
Subject(s)
Luminescence , Luminescent Measurements/methods , Nanoparticles/chemistry , Nanostructures/chemistry , Kinetics , Models, Chemical , Nanoparticles/ultrastructure , Nanostructures/ultrastructure , Particle Size , SolventsABSTRACT
The necessity to scrutinize more and more biological molecules and interactions both in solution and on the cellular level has led to an increasing demand for sensitive and specific multiplexed diagnostic analysis. Photoluminescence (PL) detection is ideally suited for multiplexed biosensing and bioimaging because it is rapid and sensitive and there is an almost unlimited choice of fluorophores that provide a large versatility of photophysical properties, including PL intensities, spectra, and lifetimes.The most frequently used technique to detect multiple parameters from a single sample is spectral (or color) multiplexing with different fluorophores, such as organic dyes, fluorescent proteins, quantum dots, or lanthanide nanoparticles and complexes. In conventional PL biosensing approaches, each fluorophore requires a distinct detection channel and excitation wavelength. This drawback can be overcome by Förster resonance energy transfer (FRET) from lanthanide donors to other fluorophore acceptors. The lanthanides' multiple and spectrally narrow emission bands over a broad spectral range can overlap with several different acceptors at once, thereby allowing FRET from one donor to multiple acceptors. The lanthanides' extremely long PL lifetimes provide two important features. First, time-gated (TG) detection allows for efficient suppression of background fluorescence from the biological environment or directly excited acceptors. Second, temporal multiplexing, for which the PL lifetimes are adjusted by the interaction with the FRET acceptor, can be used to determine specific biomolecules and/or their conformation via distinct PL decays. The high signal-to-background ratios, reproducible and precise ratiometric and homogeneous (washing-free) sensing formats, and higher-order multiplexing capabilities of lanthanide-based TG-FRET have resulted in significant advances in the analysis of biomolecular recognition. Applications range from fundamental analysis of biomolecular interactions and conformations to high-throughput and point-of-care in vitro diagnostics and DNA sequencing to advanced optical encoding, using both liquid and solid samples and in situ, in vitro, and in vivo detection with high sensitivity and selectivity.In this Account, we discuss recent advances in lanthanide-based TG-FRET for the development and application of advanced immunoassays, nucleic acid sensing, and fluorescence imaging. In addition to the different spectral and temporal multiplexing approaches, we highlight the importance of the careful design and combination of different biological, organic, and inorganic molecules and nanomaterials for an adjustable FRET donor-acceptor distance that determines the ultimate performance of the diagnostic assays and conformational sensors in their physiological environment. We conclude by sharing our vision on how progress in the development of new sensing concepts, material combinations, and instrumentation can further advance TG-FRET multiplexing and accelerate its translation into routine clinical practice and the investigation of challenging biological systems.
Subject(s)
Biosensing Techniques , Lanthanoid Series Elements , Metal Nanoparticles , Quantum Dots , Fluorescence Resonance Energy Transfer/methods , Fluorescent DyesABSTRACT
Upconversion materials have led to various breakthrough applications in solar energy conversion, imaging, and biomedicine. One key impediment is the facilitation of such processes at the molecular scale in solution where quenching effects are much more pronounced. In this work, molecular solution-state cooperative luminescence (CL) upconversion arising from a Yb excited state is explored and the mechanistic origin behind cooperative sensitisation (CS) upconversion in Yb/Tb systems is investigated. Counterintuitively, the best UC performances were obtained for Yb/Tb ratios close to parity, resulting in the brightest molecular upconversion complexes with a quantum yield of 2.8×10-6 at a low laser power density of 2.86â W cm-2 .
ABSTRACT
Introduction: Histone modifying enzymes include several classes of enzymes that are responsible for various post-translational modifications of histones such as methylation and acetylation. They are important epigenetic factors, which may involve several diseases and so their assay, as well as screening of their inhibitors, are of great importance. Herein, a bioassay based on terbium-to-quantum dot (Tb-to-QD) time-resolved Förster resonance energy transfer (TR-FRET) was developed for monitoring the activity of G9a, the euchromatic histone-lysine N-methyltransferase 2. Overexpression of G9a has been reported in some cancers such as ovarian carcinoma, lung cancer, multiple myeloma and brain cancer. Thus, inhibition of this enzyme is important for therapeutic purposes. Methods: In this assay, a biotinylated peptide was used as a G9a substrate in conjugation with streptavidin-coated ZnS/CdSe QD as FRET acceptor, and an anti-mark antibody labeled with Tb as a donor. Time-resolved fluorescence was used for measuring FRET ratios. Results: We examined three QDs, with emission wavelengths of 605, 655 and 705 nm, as FRET acceptors and investigated FRET efficiency between the Tb complex and each of them. Since the maximum FRET efficiency was obtained for Tb to QD705 (more than 50%), this pair was exploited for designing the enzyme assay. We showed that the method has excellent sensitivity and selectivity for the determination of G9a at concentrations as low as 20 pM. Furthermore, the designed assay was applied for screening of an enzyme inhibitor, S-(5'-Adenosyl)-L-homocysteine (SAH). Conclusion: It was shown that Tb-to-QD FRET is an outstanding platform for developing a homogenous assay for the G9a enzyme and its inhibitors. The obtained results confirmed that this assay was quite sensitive and could be used in the field of inhibitor screening.
ABSTRACT
Isothermal nucleic acid amplification strategies have been combined with nanotechnology for advanced biosensing, material design, and biomedical applications. However, merging phenomena and materials of different nanoscales with the aim of exploiting all their benefits at once has remained a challenging endeavor. Here, we exemplify the various problems one can encounter when combining the nanodimensions of lanthanide complexes (â¼2 nm), Förster resonance energy transfer (FRET, â¼5 nm), quantum dots (QDs, â¼20 nm), and rolling circle amplification (RCA, â¼250 nm) into a single microRNA biosensor and how these challenges can be overcome. Six different approaches, including simple FRET-RCA, enzyme-digesting FRET-RCA, and FRET-hyperbranched-RCA were investigated. We demonstrated specific miR-21 detection with 80 fM limit of detection and multiplexing capability with FRET from a Tb complex to different QDs. The detailed view on the various complex multi-nanodimensional assay systems elucidated the limited clinical translation of such sophisticated multicomponent nanobiosensors.