We are developing new and useful ways to prepare bio-inorganic conjugates of highly luminescent semiconductor quantum nanocrystals (Quantum Dots; QDs) and proteins for use in biosensing applications. Conjugate assembly isdriven by electrostatic interaction of negatively charged QD surfaces with positively charged proteins or protein subdomains. Conjugates retain the properties of both starting materials, i.e., biological activity of the proteins and optical characteristics of the QDs. We have used these hybrid bio-inorganic conjugates as tracking reagents in fluoroimmunoassays.

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.

Colloidal semiconductor nanocrystals (quantum dots, QDs), such as CdSe-ZnS core-shell, are highly luminescent and stable inorganic fluorophores that represent a promising alternative to organic dyes for a variety of biotechnological applications. They show size-tunable narrow photoluminescence spectra spanning nearly the full visible region of the optical spectrum for QDs with CdSe cores. We have developed several approaches to conjugate either one type or a combination of biologically distinct proteins to CdSe-ZnS core-shell QDs rendered water-soluble by surface ligation with dihydrolipoic acid (DHLA) groups. QD-protein conjugates prepared using these approaches were found to exhibit high specificity and stability in immunoassays and in Förster resonance energy transfer (FRET) assays as well as in prototype QD bioconjugate sensors. Tunable QD emission over a wide range of wavelengths permitted effective tuning of the degree of energy overlap between the QD donor and an acceptor dye, allowing control over the rate of FRET. Additionally, we have used these QD-bioconjugates in live cell labeling. These hybrid bioinorganic conjugates represent a promising tool for use in many biotechnological applications.

Multiple copies ( approximately 20) of Escherichia coli maltose binding protein (MBP) were coordinated to luminescent semiconductor quantum dots (QDs) via a C-terminal oligohistidine segment. The MBP was labeled with a sulfo-N-hydroxysuccinimide-activated photochromic BIPS molecule (1',3-dihydro-1'-(2-carboxyethyl)-3,3-dimethyl-6-nitrospiro[2H-1-benzopyran-2,2'-(2H)-indoline]) at two different dye-to-MBP ratios; D/P = 1 and 5. The ability of MBP-BIPS to modulate QD photoluminescence was tested by switching BIPS from the colorless spiropyran (SP) to the colored merocyanine (MC) using white light (>500 nm) or UV light ( approximately 365 nm), respectively. QDs surrounded by MBP-BIPS with D/P = 1 were quenched on average approximately 25% with consecutive repeated switches, while QDs surrounded by MBP-BIPS with D/P = 5 were quenched approximately 60%. This result suggests a possible use of BIPS-labeled proteins in QD-based nanostructures as part of a threshold switch or other biosensing device.

We report a simple and versatile approach for the conjugation of luminescent CdSe–ZnS core-shell quantum dots (QDs) to proteins through coordination of engineered C-terminal oligohistidine sequences. Several histidine tail containing proteins were self-assembled onto the QD surface using this method. A recombinant antibody specific for the high explosive 2,4,6-trinitrotoluene (TNT) was conjugated to QDs through a carboxy terminal histidine tail and the bioconjugate used to detect TNT by competitive immunoassay. TNT was detected over the range of 10 μg/ml down to 41 ng/ml using the scFv conjugated to QDs. These results open up the possibility to conjugate luminescent QDs to a whole range of proteins to form QD bioconjugates that can be effectively used in bio-oriented applications, such as sensing, imaging, immunoassay and other diagnostics.

ABSTRACT Reagentless bio‐sensing proteins represent a promising technology being developed for sensitive chemical detection and analysis. The majority of these sensor proteins incorporate environmentally sensitive UV‐excited fluorescent dyes as reporter probes. In this report, we describe conjugation of the commercially available visible wavelength‐excitable polymethine cyanine dye Cy3 (λmax abs = 556 nm; λmax em = 567 nm) to several single‐cysteine variants of maltose binding protein (MBP), followed by evaluation of each of these labeled proteins as potential reagentless sensors for maltose. Four of six MBP variants labeled with the Cy3 probe functioned successfully in a homogenous reagentless sensing mode, and yielded maltose dissociation constants ranging from 74 µM to 3.8 mM. Cy3 and other cyanine dye derivatives may be useful as long‐wavelength excitable reporter groups in other reagentless sensor designs.