RESUMEN
The unique properties provided by hybrid semiconductor quantum dot (QD) bioconjugates continue to stimulate interest for many applications ranging from biosensing to energy harvesting. Understanding both the structure and function of these composite materials is an important component in their development. Here, we compare the architecture that results from using two common self-assembly chemistries to attach DNA to QDs. DNA modified to display either a terminal biotin or an oligohistidine peptidyl sequence was assembled to streptavidin/amphiphilic polymer- or PEG-functionalized QDs, respectively. A series of complementary acceptor dye-labeled DNA were hybridized to different positions on the DNA in each QD configuration and the separation distances between the QD donor and each dye-acceptor probed with Förster resonance energy transfer (FRET). The polyhistidine self-assembly yielded QD-DNA bioconjugates where predicted and experimental separation distances matched reasonably well. Although displaying efficient FRET, data from QD-DNA bioconjugates assembled using biotin-streptavidin chemistry did not match any predicted separation distances. Modeling based upon known QD and DNA structures along with the linkage chemistry and FRET-derived distances was used to simulate each QD-DNA structure and provide insight into the underlying architecture. Although displaying some rotational freedom, the DNA modified with the polyhistidine assembles to the QD with its structure extended out from the QD-PEG surface as predicted. In contrast, the random orientation of streptavidin on the QD surface resulted in DNA with a wide variety of possible orientations relative to the QD which cannot be controlled during assembly. These results suggest that if a particular QD biocomposite structure is desired, for example, random versus oriented, the type of bioconjugation chemistry utilized will be a key influencing factor.
Asunto(s)
ADN/química , Nanocompuestos/química , Puntos Cuánticos , Secuencia de Bases , Biotina/metabolismo , ADN/genética , ADN/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Histidina/metabolismo , Modelos Moleculares , Conformación Molecular , Fragmentos de Péptidos/química , Polietilenglicoles/química , Estreptavidina/químicaRESUMEN
Biosensors function by reversibly linking bioreceptor-target analyte binding with closely integrated signal generation and can either continuously monitor analyte concentrations or be returned to baseline readout values by removal of analyte. We present an approach for producing fully reversible, reagentless, self-assembling biosensors on surfaces. In the prototype biosensor, quencher-dye-labeled biotin-linked E. coli maltose binding protein (MBP) bound in a specific orientation to a NeutrAvidin-coated surface is employed as a bioreceptor. To complete sensor formation, a modular tether arm consisting of a flexible biotinylated DNA oligonucleotide, a fluorescence resonance energy-transfer (FRET) donor dye, and a distal beta-cyclodextrin (beta-CD) analyte analogue is bound in an equimolar amount to the same surface by means of DNA-directed immobilization. After self-assembly, a baseline level of FRET quenching is observed due to specific interaction between the beta-CD of the flexible tether arm and the sugar binding site of MBP, which brings the two dyes into proximity. Addition of the target analyte, the nutrient maltose, displaces the linked beta-CD-dye of the DNA-based tether arm, and a concentration-dependent change in FRET results. Biosensor sensitivity and dynamic range can be controlled by either using MBP variants having different binding constants or by binding of modulator DNA oligonucleotides that are complementary to the flexible DNA tether. The sensor can be regenerated and returned to baseline quenching levels by washing away analyte. A complex set of interactions apparently exists on the sensing surface that may contribute to sensor behavior and range. This approach may represent a general way to assemble a wide range of useful biosensors.
Asunto(s)
Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Secuencia de Bases , ADN/química , ADN/genética , Proteínas de Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Datos de Secuencia Molecular , Proteínas de Unión Periplasmáticas/metabolismo , Sensibilidad y Especificidad , TemperaturaRESUMEN
Quantum dots (QDs) have the potential to simplify the performance of multiplexed analysis. In this work, we prepared bioinorganic conjugates made with highly luminescent semiconductor nanocrystals (CdSe-ZnS core-shell QDs) and antibodies to perform multiplexed fluoroimmunoassays. Sandwich immunoassays for the detection of cholera toxin, ricin, shiga-like toxin 1, and staphylococcal enterotoxin B were performed simultaneously in single wells of a microtiter plate. Initially the assay performance for the detection of each toxin was examined. We then demonstrated the simultaneous detection of the four toxins from a single sample probed with a mixture of all four QD-antibody reagents. Using a simple linear equation-based algorithm, it was possible to deconvolute the signal from mixed toxin samples, which allowed quantitation of all four toxins simultaneously.