J. Matthew Mauro

Long-term multiple color imaging of live cells using quantum dot bioconjugates

Luminescent quantum dots (QDs)—semiconductor nanocrystals—are a promising alternative to organic dyes for fluorescence-based applications. We have developed procedures for using QDs to label live cells and have demonstrated their use for long-term multicolor imaging of live cells. The two approaches presented are (i) endocytic uptake of QDs and (ii) selective labeling of cell surface proteins with QDs conjugated to antibodies. Live cells labeled using these approaches were used for long-term multicolor imaging. The cells remained stably labeled for over a week as they grew and developed. These approaches should permit the simultaneous study of multiple cells over long periods of time as they proceed through growth and development.

Protection against experimental bubonic and pneumonic plague by a recombinant capsular F1-V antigen fusion protein vaccine.

The current human whole-cell vaccine is ineffective against pneumonic plague caused by typical F1 capsule positive (F1+) strains of Yersinia pestis. The authors found this vaccine to also be ineffective against F1-negative (F1-) Y. pestis strains, which have been isolated from a human case and from rodents. For these reasons, the authors developed a recombinant vaccine composed of a fusion protein of F1 with a second protective immunogen, V antigen. This vaccine protected experimental mice against pneumonic as well as bubonic plague produced by either an F1+ or F1- strain of Y. pestis, gave better protection than F1 or V alone against the F1+ strain, and may provide the basis for an improved human plague vaccine.

Phage-displayed peptides as biosensor reagents

This study investigated the potential to utilize phage‐displayed peptides as reagents in sensor applications. A library of random 12‐mers displayed on phage was panned against staphylococcal enterotoxin B (SEB), a causative agent of food poisoning. Nine SEB binding phage clones were isolated, all of which share the consensus sequence Trp His Lys at their amino terminus. Binding of several of these phage was shown to be inhibited when they were assayed in a competitive enzyme‐linked immunosorbent assay (ELISA) format with synthesized peptide corresponding to the peptide‐encoding region of one of the clones. Whole phage were labeled with the dye Cy5, and incorporated into fluoroimmunoassays. Labeled phage were able to detect SEB down to a concentration of 1.4 ng/well in a fluorescence‐based immunoassay. When incorporated into an automated fluorescence‐based sensing assay, Cy5‐labeled phage bound to probes coated with SEB generated a robust signal of about 10,000 pA, vs a signal of 1000 pA using a control fiber coated with streptavidin. These results demonstrate the potential for development of phage‐based sensor reagents. Copyright

A Fluorescence Resonance Energy Transfer Sensor Based on Maltose Binding Protein

A fluorescence resonance energy-transfer (FRET) sensing system for maltose based on E. coli maltose binding protein (MBP) is demonstrated. The FRET donor portion of the sensing system consists of MBP modified with long wavelength-excitable cyanine dyes (Cy3 or Cy3.5). The novel acceptor portion of the sensor consists of beta-cyclodextrin (beta-CD) modified with either the cyanine dye Cy5 or the dark quencher QSY9. Binding of the modified beta-CD to dye-conjugated MBP results in assembly of the FRET complex. Added maltose displaces the beta-CD-dye adduct and disrupts the FRET complex, resulting in a direct change in fluorescence of the donor moiety. In the use of these FRET pairs, MBP dissociation values for maltose were estimated (0.14-2.90 microM). Maltose limits of detection were in the 50-100 nm range.

Fluoroimmunoassays Using Antibody-Conjugated Quantum Dots

Luminescent colloidal semiconductor nanocrystals (quantum dots) are robust inorganic fluoro phores that have the potential to circumvent some of the functional limitations encountered by organic dyes in sensing and biotechnological applications. Quantum dots exhibit size-dependent tunable, narrow fluorescence emission spectra that span the visible spectrum and have broad absorption spectra. This allows simultaneous excitation of several particle sizes at a single wavelength with emission at multiple wavelengths. Quantum dots also provide a high-resistance threshold to chemical degradation and photodegradation. We have developed a conjugation strategy for the attachment of antibodies to quantum dots based on electrostatic interactions between negatively charged dihydrolipoic acid (DHLA)-capped CdSe-ZnS core-shell quantum dots and positively charged proteins (natural or engineered) that serve to bridge the quantum dot and antibody. This chapter details the materials and methods for synthesis of the DHLA-capped CdSe-ZnS core-shell quantum dots, the construction and preparation of recombinant proteins, the conjugation of antibodies to quantum dots, and the use of antibody-coated quantum dots in a fluoroimmunoassay.

Luminescent Quantum Dot-Bioconjugates in Immunoassays, FRET, Biosensing, and Imaging Applications

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.

Colloidal Semiconductor Quantum Dot Conjugates in Biosensing

Publisher Summary This chapter reviews the progress made in bio-related applications of luminescent colloidal quantum dots (QDs). The material reviewed represents only the prologue of an unfolding story, as quantum dots are a relatively recent discovery and their biological applications are newer still. Nonetheless, a significant body of research literature exists pointing the way toward future advances. We begin with a basic introduction to quantum dots, including their synthesis and some characteristic physical properties, followed by a review of bio-related work involving semiconductor nanocrystals published to date. Work involving the preparation and use of QD-protein conjugates in cellular imaging, quantitative immunoassays, and in early-stage energy transfer applications is reviewed, in addition to the uses of QD-DNA conjugates as nanoscale building blocks. A listing of early patents in this area is also included for those who contemplate utilizing these materials in the commercial arena. Advantages and limitations in bio-related applications are discussed based on the current state-of-the-art in QD technology.

A reagentless electrochemical biosensor based on a protein scaffoldElectronic supplementary information (ESI) available: details regarding protein engineering and purification. See http://www.rsc.org/suppdata/cc/b2/b209452e/

Apo-myoglobin, labeled with the environmentally sensitive redox probe RuII(NH3)4(1,10-phenanthroline-5-maleimide)2+, was immobilized onto gold electrodes modified with 11-mercaptoundecanoic acid and subsequently labeled with biotin; avidin binding to the immobilized biotin was specifically and quantitatively detected by a change in cyclic voltammetry of the co-immobilized probe.

Use of a Cyanine Dye as a Reporter Probe in Reagentless Maltose Sensors Based on E. coli Maltose Binding Protein

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.

Bioconjugates of luminescent CdSe-ZnS quantum dots with an engineered two-domain protein G for use in fluoroimmunoassays

Colloidal semiconductor quantum dots (QDs) seem suitable for labeling certain biomolecules for use in fluorescent tagging applications, such as fluoro-immunoassays. Compared to organic dye labels, Qds are resistant to photo-degradation, and these luminescent nanoparticles have size-dependent emission spectra spanning a wide range of wavelengths in the visible and near IR. We previously described an electrostatic self-assembly approach for conjugating highly luminescent colloidal CdSe-ZnS core-shell Qds with engineered two-domain recombinant proteins. Here we describe the application of this approach to prepare QD conjugates with the (Beta) 2 immunoglobin G (IgG) binding domain of streptococcal protein G (PG) appended with a basic lucine zipper attachment domain (PG-zb). We also demonstrate that the QD/PG conjugates retain their ability to bind IgG antibodies, and that a specific antibody coupled to QD via the PG functional domain efficiently binds its antigen. These preliminary results indicate that electrostatically self-assembled QD/PG-zb/IgG bioconjugates can be used in fluoro-immunoassays.