Quantum Dot Lipase Biosensor Utilizing a Custom-Synthesized Peptidyl-Ester Substrate (original) (raw)
Related papers
Analytical Chemistry, 2007
The paper describes the development and characterization of analytical properties of quantum dot-based probes for enzymatic activity and for screening enzyme inhibitors. The luminescent probes are based on fluorescence resonance energy transfer (FRET) between luminescent quantum dots that serve as donors and rhodamine acceptors that are immobilized to the surface of the quantum dots through peptide linkers. Peptide-coated CdSe/ZnS quantum dots were prepared using a one-step ligand exchange process in which RGDC peptide molecules replace trioctylphosphine oxide (TOPO) molecules as the capping ligands of the quantum dots. The peptide molecules were bound to the surface of the CdSe/ZnS quantum dots through the thiol group of the peptide cysteine residue. The peptide-coated quantum dots were labeled with rhodamine to form the FRET probes. The emission quantum yield of the quantum dot FRET probes was 4-fold lower than the emission quantum yield of TOPO-capped quantum dots. However, the quantum dot FRET probes were sufficiently bright to enable quantitative enzyme and enzyme inhibition assays. The probes were used first to test the enzymatic activity of trypsin in solution based on FRET signal changes of the quantum dot-based enzymatic probes in the presence of proteolytic enzymes. For example, exposure of the quantum dot FRET probes to 500 µg/mL trypsin for 15 min resulted in 60% increase in the photoluminescence of the quantum dots and a corresponding decrease in the emission of the rhodamine molecules. These changes resulted from the release of rhodamine molecules from the surface of the quantum dots due to enzymatic cleavage of the peptide molecules. The quantum dot FRET-based probes were used to monitor the enzymatic activity of trypsin and to screen trypsin inhibitors for their inhibition efficiency.
Quantum dot–NBD–liposome luminescent probes for monitoring phospholipase A2 activity
Analytical and Bioanalytical Chemistry, 2013
In this paper we describe the fabrication and characterization of new liposome encapsulated quantum dot-fluorescence resonance energy transfer (FRET)-based probes for monitoring the enzymatic activity of phospholipase A 2. To fabricate the probes, luminescent CdSe/ZnS quantum dots capped with trioctylphosphine oxide (TOPO) ligands were incorporated into the lipid bilayer of unilamellar liposomes with an average diameter of approximately 100 nm. Incorporating TOPO capped quantum dots in liposomes enabled their use in aqueous solution while maintaining their hydrophobicity an d ex cel l e nt photophysical properties. The phospholipid bilayer was labeled with the fluorophore NBD C 6-HPC (2-(6-(7nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexa decanoyl-sn-glycero-3-phosphocholine). The luminescent quantum dots acted as FRET donors and the NBD dye molecules acted as FRET acceptors. The probe response was based on FRET interactions between the quantum dots and the NBD dye molecules. The NBD dye molecules were cleaved and released to the solution in the presence of the enzyme phospholipase A 2. This led to an increase of the luminescence of the quantum dots and to a corresponding decrease in the fluorescence of the NBD molecules, because of a decrease in FRET efficiency between the quantum dots and the NBD dye molecules. Because the quantum dots were not attached covalently to the phospholipids, they did not hinder the enzyme activity as a result of steric effects. The probes were able to detect amounts of phospholipase A 2 as low as 0.0075 U mL −1 and to monitor enzyme activity in real time. The probes were also used to screen phospholipase A 2 inhibitors. For example, we found that the inhibition efficiency of MJ33 (1-hexadecyl-3-(trifluoroethyl)-snglycero-2-phosphomethanol) was higher than that of OBAA (3-(4-octadecyl)benzoylacrylic acid).
Quantum Dot Peptide Biosensors for Monitoring Caspase 3 Proteolysis and Calcium Ions
ACS Nano, 2010
The nanoscale size and unique optical properties of semiconductor quantum dots (QDs) have made them attractive as central photoluminescent scaffolds for a variety of biosensing platforms. In this report we functionalize QDs with dye-labeled peptides using two different linkage chemistries to yield Förster resonance energy transfer (FRET)-based sensors capable of monitoring either enzymatic activity or ionic presence. The first sensor targets the proteolytic activity of caspase 3, a key downstream effector of apoptosis. This QD conjugate utilized carbodiimide chemistry to covalently link dye-labeled peptide substrates to the terminal carboxyl groups on the QD's surface hydrophilic ligands in a quantitative manner. Caspase 3 cleaved the peptide substrate and disrupted QD donor-dye acceptor FRET providing signal transduction of enzymatic activity and allowing derivation of relevant Michaelis؊Menten kinetic descriptors. The second sensor was designed to monitor Ca 2؉ ions that are ubiquitous in many biological processes. For this sensor, Cu ؉ -catalyzed [3 ؉ 2] azide؊alkyne cycloaddition was exploited to attach a recently developed azide-functionalized CalciumRuby-Cl indicator dye to a cognate alkyne group present on the terminus of a modified peptide. The labeled peptide also expressed a polyhistidine sequence, which facilitated its subsequent metal-affinity coordination to the QD surface establishing the final FRET sensing construct. Adding exogenous Ca 2؉ to the sensor solution increased the dyes fluorescence, altering the donor؊acceptor emission ratio and manifested a dissociation constant similar to that of the native dye. These results highlight the potential for combining peptides with QDs using different chemistries to create sensors for monitoring chemical compounds and biological processes.
Nanoparticle modified QCM-based sensor for lipase activity determination
Analytical Methods, 2013
A highly sensitive lipase activity sensor was developed and tested. It is based on the application of SiO 2 nanoparticle loaded olive oil as a lipase substrate, deposited on a QCM crystal. The heavier nanoparticles' release during the substrate enzymatic degradation causes a QCM frequency response enhancement proportional to the nanoparticle/substrate mass ratio.
Proceedings of the National Academy of Sciences, 2004
The first generation of luminescent semiconductor quantum dot (QD)-based hybrid inorganic biomaterials and sensors is now being developed. It is crucial to understand how bioreceptors, especially proteins, interact with these inorganic nanomaterials. As a model system for study, we use Rhodamine red-labeled engineered variants of Escherichia coli maltose-binding protein (MBP) coordinated to the surface of 555-nm emitting CdSe-ZnS core–shell QDs. Fluorescence resonance energy transfer studies were performed to determine the distance from each of six unique MBP-Rhodamine red dye-acceptor locations to the center of the energy-donating QD. In a strategy analogous to a nanoscale global positioning system determination, we use the intraassembly distances determined from the fluorescence resonance energy transfer measurements, the MBP crystallographic coordinates, and a least-squares approach to determine the orientation of the MBP relative to the QD surface. Results indicate that MBP has ...
Biosensors
Bioluminescence resonance energy transfer (BRET) techniques offer a high degree of sensitivity, reliability and ease of use for their application to sensing biomolecules. BRET is a distance dependent, non-radiative energy transfer, which uses a bioluminescent protein to excite an acceptor through the resonance energy transfer. A BRET sensor can quickly detect the change of a target biomolecule quantitatively without an external electromagnetic field, e.g., UV light, which normally can damage tissue. Having been developed quite recently, this technique has evolved rapidly. Here, different bioluminescent proteins have been reviewed. In addition to a multitude of bioluminescent proteins, this manuscript focuses on the recent development of BRET sensors by utilizing quantum dots. The special size-dependent properties of quantum dots have made the BRET sensing technique attractive for the real-time monitoring of the changes of target molecules and bioimaging in vivo. This review offers a...
Quantum dots as resonance energy transfer acceptors for monitoring biological interactions
Biophotonics and New Therapy Frontiers, 2006
Due to their extraordinary photophysical properties CdSe/ZnS core/shell nanocrystals (quantum dots) are excellent luminescence dyes for fluorescence resonance energy transfer (FRET) systems. By using a supramolecular lanthanide complex with central terbium cation as energy donor, we show that commercially available biocompatible biotinilated quantum dots are excellent energy acceptors in a time-resolved FRET fluoroimmunoassay (FRET-FIA) using streptavidin-biotin binding as biological recognition process. The efficient energy transfer is demonstrated by quantum dot emission sensitization and a thousandfold increase of the nanocrystal luminescence decay time. A Förster Radius of 90 Å and a picomolar detection limit were achieved in quantum dot borate buffer. Regarding biological applications the influence of bovine serum albumin (BSA) and sodium azide (a frequently used preservative) to the luminescence behaviour of our FRET-system is reported.
2010
CdSe/ZnS core/shell quantum dots (QDs) are functionalized with mercaptoundecanoic acid (MUA) and subsequently covered with poly-L-lysine (PLL) as the template for the formation of the silica outer shell. This nanocomposite is used as a transduction and stabilization system for optical biosensor development. The covalent immobilization of the enzyme acetylcholinesterase from Drosophila melanogaster (AChE) during the formation of the biomimetically synthesized silica is used here as a model, relatively unstable enzyme, as a proof of principle. The enzyme is successfully immobilized onto the QDs and then stabilized by the PLL capping and the subsequent formation of the outer nanoporous silica thin shell, giving rise to the QD/AChE/PLL/silica biosensor. It is shown that the poly-L-lysine templated silica outer shell does not modify the optical properties of the quantum dots, while it protects the enzyme from unfolding and denaturation. The small pores of the silica shell allow for the free diffusion of the analyte to the active center of the enzyme, while it does not allow for the proteases to reach the enzyme. The response of the QD/AChE/PLL/silica nano-biosensor to its substrate, acetylcholine chloride, is evaluated by monitoring the changes in the QDs' photoluminescence which are related to the changes in pH. These pH changes of the surrounding environment of the QDs are induced by the enzymatic reaction, and are associated with the analyte concentration in the solution. The biodetection system proposed is shown to be stable with a storage lifetime of more than 2 months. The data presented provides the grounds for the application of this nanostructured biosensor for the detection of AChE inhibitors.
Hydrolytic enzymes conjugated to quantum dots mostly retain whole catalytic activity
Biochimica et Biophysica Acta (BBA) - General Subjects, 2014
Tagging a luminescent quantum dot (QD) with a biological like enzyme (Enz) creates valueadded entities like quantum dot-enzyme bioconjugates (QDEnzBio) that find utility as sensors to detect glucose or beacons to track enzymes in vivo. For such applications, it is imperative that the enzyme remains catalytically active while the QD is luminescent in the bioconjugate. A critical feature that dictates this is the QD-Enz linkage chemistry. Previously such linkages have put constraints on polypeptide chain dynamics or hindered substrate diffusion to active site, seriously undermining enzyme catalytic activity. In this work we address this issue using Avidin-Biotin linkage chemistry together with a flexible spacer to conjugate enzyme to QD.
Protein–Conjugated Quantum Dots Interface: Binding Kinetics and Label-Free Lipid Detection
Analytical Chemistry, 2014
We propose a label-free biosensor platform to investigate the binding kinetics using antigen−antibody interaction via electrochemical and surface plasmon resonance (SPR) techniques. The L-cysteine in situ capped cadmium sulfide (CdS; size < 7 nm) quantum dots (QDs) selfassembled on gold (Au) coated glass electrode have been covalently functionalized with apolipoprotein B-100 antibodies (AAB). This protein conjugated QDs-based electrode (AAB/ CysCdS/Au) has been used to detect lipid (low density lipoprotein, LDL) biomolecules. The electrochemical impedimetric response of the AAB/CysCdS/Au biosensor shows higher sensitivity (32.8 kΩ μM −1 /cm 2 ) in the detection range, 5−120 mg/dL. Besides this, efforts have been made to investigate the kinetics of antigen−antibody interactions at the CysCdS surface. The label-free SPR response of AAB/CysCdS/Au biosensor exhibits highly specific interaction to protein (LDL) with association constant of 33.4 kM −1 s −1 indicating higher affinity toward LDL biomolecules and a dissociation constant of 0.896 ms −1 . The results of these studies prove the efficacy of the CysCdS-Au platform as a high throughput compact biosensing device for investigating biomolecular interactions.