QD-based Fluorescence Resonance Energy Transfer Research Articles

High-fidelity CRISPR/Cas13a trans cleavage-driven assembly of single quantum dot nanosensor for ultrasensitive detection of long noncoding RNAs in clinical breast tissues

Long noncoding RNAs (lncRNAs) act as critical regulators in various cellular processes, and their dysfunction is implicated in carcinogenesis. Herein, we demonstrate high-fidelity CRISPR/Cas13a trans cleavage-driven assembly of single quantum dot (QD) nanosensor for ultrasensitive detection of long noncoding RNAs in clinical tissues. The presence of lncRNA can activate Cas13a/crRNA to collaterally cleave the substrate probes, producing a T7 promoter fragment that can initiate subsequent transcription amplification to generate efficient fluorescence resonance energy transfer (FRET). Taking advantage of excellent specificity of high-fidelity CRISPR/Cas13a system, high efficiency of transcription amplification, and near-zero background of single QD-based FRET, this nanosensor can achieve a detection limit of 1.65 aM, and it can differentiate target lncRNA from its mismatched members with single-base resolution. Moreover, it can measure lncRNA at the single-cell level, distinguish different subtypes of breast cancers, and assess the breast cancer progression. Notably, due to the programmability of crRNAs, this nanosensor can be extended to detect other nucleic acids (e.g., SARS-CoV-2 RNA, circRNA, miRNA, piRNA, and 16S rRNA) by simply altering the spacer region of crRNA, with great potential in lncRNAs-related molecular diagnostics.

A High-Quality CdSe/CdS/ZnS Quantum-Dot-Based FRET Aptasensor for the Simultaneous Detection of Two Different Alzheimer's Disease Core Biomarkers.

The simultaneous detection of two different biomarkers for the point-of-care diagnosis of major diseases, such as Alzheimer's disease (AD), is greatly challenging. Due to the outstanding photoluminescence (PL) properties of quantum dots (QDs), a high-quality CdSe/CdS/ZnS QD-based fluorescence resonance energy transfer (FRET) aptasensor for simultaneously monitoring the amyloid-β oligomers (AβO) and tau protein was proposed. By engineering the interior inorganic structure and inorganic-organic interface, water-soluble dual-color CdSe/CdS/ZnS QDs with a near-unity PL quantum yield (>90%) and mono-exponential PL decay dynamics were generated. The π-π stacking and hydrogen bond interaction between the aptamer-functionalized dual-color QDs and gold nanorods@polydopamine (Au NRs@PDA) nanoparticles resulted in significant fluorescence quenching of the QDs through FRET. Upon the incorporation of the AβO and tau protein, the fluorescence recovery of the QDs-DNA/Au NRs@PDA assembly was attained, providing the possibility of simultaneously assaying the two types of AD core biomarkers. The lower detection limits of 50 pM for AβO and 20 pM for the tau protein could be ascribed to the distinguishable and robust fluorescence of QDs and broad spectral absorption of Au NRs@PDA. The sensing strategy serves as a viable platform for the simultaneously monitoring of the core biomarkers for AD and other major diseases.

Hydroxymethylation-Specific Ligation-Mediated Single Quantum Dot-Based Nanosensors for Sensitive Detection of 5-Hydroxymethylcytosine in Cancer Cells.

5-Hydroxymethylcytosine (5hmC) modification is a key epigenetic regulator of cellular processes in mammalian cells, and its misregulation may lead to various diseases. Herein, we develop a hydroxymethylation-specific ligation-mediated single quantum dot (QD)-based fluorescence resonance energy transfer (FRET) nanosensor for sensitive quantification of 5hmC modification in cancer cells. We design a Cy5-modified signal probe and a biotinylated capture probe for the recognition of specific 5hmC-containing genes. 5hmC in target DNA can be selectively converted by T4 β-glucosyltransferase to produce a glycosyl-modified 5hmC, which cannot be cleaved by methylation-insensitive restriction enzyme MspI. The glycosylated 5hmC DNA may act as a template to ligate a signal probe and a capture probe, initiating hydroxymethylation-specific ligation to generate large amounts of biotin-/Cy5-modified single-stranded DNAs (ssDNAs). The assembly of biotin-/Cy5-modified ssDNAs onto a single QD through streptavidin-biotin interaction results in FRET and consequently the generation of a Cy5 signal. The nanosensor is very simple without the need for bisulfite treatment, radioactive reagents, and 5hmC-specific antibodies. Owing to excellent specificity and high amplification efficiency of hydroxymethylation-specific ligation and near-zero background of a single QD-based FRET, this nanosensor can quantify 5hmC DNA with a limit of detection of 33.61 aM and a wider linear range of 7 orders of magnitude, and it may discriminate the single-nucleotide difference among 5hmC, 5-methylcytosine, and unmodified cytosine. Moreover, this nanosensor can distinguish as low as a 0.001% 5hmC DNA in complex mixtures, and it can monitor the cellular 5hmC level and discriminate cancer cells from normal cells, holding great potential in biomedical research and clinical diagnostics.

The Current Status and Future Outlook of Quantum Dot-Based Biosensors for Plant Virus Detection.

Enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), widely used for the detection of plant viruses, are not easily performed, resulting in a demand for an innovative and more efficient diagnostic method. This paper summarizes the characteristics and research trends of biosensors focusing on the physicochemical properties of both interface elements and bioconjugates. In particular, the topological and photophysical properties of quantum dots (QDs) are discussed, along with QD-based biosensors and their practical applications. The QD-based Fluorescence Resonance Energy Transfer (FRET) genosensor, most widely used in the biomolecule detection fields, and QD-based nanosensor for Rev-RRE interaction assay are presented as examples. In recent years, QD-based biosensors have emerged as a new class of sensor and are expected to open opportunities in plant virus detection, but as yet there have been very few practical applications (Table 3). In this article, the details of those cases and their significance for the future of plant virus detection will be discussed.

A single quantum dot-based nanosensor for the signal-on detection of DNA methyltransferase.

We develop a single quantum dot (QD)-based nanosensor for the signal-on detection of DNA methyltransferase (MTase). By integration of single-molecule counting with the QD-based fluorescence resonance energy transfer (FRET), the proposed nanosensor can sensitively detect DNA MTase with a detection limit of as low as 0.002 U mL-1, and it can be further applied for inhibitor screening and accurate detection of DNA MTase in complex biological samples.

FRET from core and core–shell quantum dots to laser dye: A comparative investigation

A remarkable progress has been made in the recent past in fluorescence resonance energy transfer (FRET) studies using semiconductor quantum dots (QDs) and application of QD-based FRET to probe specific biological processes. Here we report a systematic study of FRET from CdSe (core) and CdSe/ZnS (core–shell) quantum dots (donor) to sulforhodamine B (SRB) dye (acceptor) using steadystate and time resolved fluorescence spectroscopy. The results obtained demonstrate occurrence of efficient energy transfer from QDs to dye and are discussed in the light of the Förster theory. Time resolved decay curves of QDs obtained in the presence and absence of sulforhodamine B dye are analyzed. The values of spectral overlap, Förster distance and transfer efficiency have been determined using steady-state and time-resolved fluorescence spectroscopic data. It is observed that the transfer efficiency does not follow a linear dependence on spectral overlap integral and quantum yield of donor as predicted by the Förster theory for molecules. This nonlinear dependence of transfer efficiency on overlap integral as well as quantum yield of donor; and the possibility of surface states and defect states of QDs causing this are discussed.

Quantum dot-based resonance energy transfer and its growing application in biology

We provide an overview of the progress made in the past few years in investigating fluorescence resonance energy transfer (FRET) using semiconductor quantum dots (QDs) and the application of QD-based FRET to probe specific biological processes. We start by providing some of the pertinent conceptual elements involved in resonance energy transfer, and then discuss why the Förster dipole-dipole mechanism applies to QD fluorophores. We then describe the unique QD photophysical properties of direct relevance to FRET and summarize the main advantages offered, along with some of the limitations encountered by QDs as exciton donors and/or acceptors. Next we describe the overall progress made and discuss a few representative examples where QD-based FRET sensing of specific biological processes has been demonstrated. We also detail some of the advances of single molecule FRET using QD-conjugates and highlight the unique information that can be extracted. We conclude by providing an assessment of where QD-based FRET investigations may be evolving in the near future.

Open Sandwich FRET Immunoassay of Estrogen Receptor β in a PDMS Microfluidic Channel

Immunoassay is a fast and cost-effective protein detection method that can be applied to clinical diagnostics and biological research. Enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and protein array chips are the most widely used immunoassay techniques for the detection of a target protein. In all of these methods, however, antibodies or antigens should be immobilized on a solid substrate. As a result, they require a long assay time for complete immunoreaction between antigens and antibodies, being limited by the diffusion kinetics. Moreover, they also require several cycles of consecutive binding and washing steps to separate free reagents from binding reagents. To overcome the problem of slow immunoreactions in conventional immunoassay techniques, we recently reported a fast and sensitive one-step immunoassay of estrogen receptor β (ERβ) using quantum dot (QD)-based fluorescence resonance energy transfer (FRET). To induce a strong FRET effect, QD was used as a fluorescence donor. FRET occurs when the electronic excitation energy of a donor chromophore is transferred to a nearby acceptor molecule via a dipole-dipole interaction between the donoracceptor pair. Conventional fluorescence dyes, which cause an appreciable overlap between the emission band of the donor and the absorption band of the acceptor, have generally been used as a donor-acceptor set. However, QD provides significant advantages over conventional fluorescence dyes, including brighter fluorescence, resistance to photobleaching, and narrow emission bands, which makes it suitable as a sensitive fluorescence donor. As a result, QD reaches a longer distance to the acceptor than conventional fluorescence dyes because of their superior optical properties. Furthermore, the QD-based FRET immunoassay of ERβ was faster (30-40 min of immunoassay time including incubation) than conventional immunoassay techniques because it does not need any solid-phase carriers and multiple washing steps for free reagent separations. In our previous work, the advantages of a QD-based FRET immunoassay in an open space were fully explored. Nonetheless, a faster reaction time and a smaller sample volume are still needed to apply this QD-based FRET immunoanalysis technique to clinical diagnostics. In the present work, a lab-on-a-chip technique, combined with the QD-based FRET immunoassay method, was used to develop a more sensitive and faster immunoanalysis technique. The lab-on-a-chip technique has several advantages over conventional bench-top techniques, such as minimal sample requirement, reduced reaction time, ease of use, improved product conversion, and reduced waste generation. In particular, the reaction time can be greatly reduced to less than a few minutes if a properly designed channel to obtain highly efficient mixing is adopted. Furthermore, only a small reaction volume is required in the lab-ona-chip analysis. An alligator-teeth-shaped poly(dimethylsiloxane) (PDMS) microfluidic channel was fabricated to obtain a fast immunoreaction between antibody and antigen under a flow condition. The detailed fabrication process for this PDMS channel has been reported elsewhere. ERβ is one of the most important breast cancer markers that are closely related with cell proliferations. The antiERβ monoclonal antibody (McAb, mouse ascites, IgM isotype; Sigma, St. Louis, MO, USA) was labeled on QD 565 using an antibody conjugation kit (Invitrogen, Eugene, OR, USA). In addition, the anti-ERβ polyclonal antibody (PcAb, rabbit immune serum, IgG; Biogenex, San Ramon, CA, USA) was labeled with AF 568 protein labeling kits (Invitrogen, Eugene, OR, USA). The formation of an open sandwich immunocomplex and its energy transfer process from QD-McAb to AF-PcAb are illustrated in Figure 1(a).

Quantum Dot−Fluorescent Protein Pairs as Novel Fluorescence Resonance Energy Transfer Probes

Fluorescence resonance energy transfer (FRET) characteristics, including the efficiency, donor-acceptor distance, and binding strength of six fluorescent protein (FP)-quantum dot (QD) pairs were quantified, demonstrating that FPs are efficient acceptors for QD donors with up to 90% quenching of QD fluorescence and that polyhistidine coordination to QD core-shell surface is a straightforward and effective means of conjugating proteins to commercially available QDs. This provides a novel approach to developing QD-based FRET probes for biomedical applications.

Quantifying RNA−Peptide Interaction by Single-quantum Dot-Based Nanosensor: An Approach for Drug Screening

The sequence-specific RRE RNA-Rev binding is essential for HIV-1 replication and provides a useful in vitro system for real-time evaluating the inhibitory effect of drugs on the RRE-Rev interaction. The rapid and sensitive detection of RRE-Rev interaction in complex biological systems represents a fundamental challenge. Here we report the development of a single-quantum-dot (QD)-based nanosensor for sensitively quantifying Rev peptide-RRE interaction and characterizing the potential inhibitors by virtue of single-molecule detection and QD-based fluorescence resonance energy transfer (FRET). We demonstrate that the stoichiometry of Rev peptide binding to RRE can be accurately determined by using this single-QD-based nanosensor. Importantly, this single-QD-based nanosensor can sensitively quantify the inhibitory efficacy of proflavin on the Rev peptide-RRE binding, even in the presence of substantial levels of interference fluorescence from high-concentration proflavin, which usually prevents the discrimination of FRET signals in ensemble measurements. The application of this nanosensor in the screening of libraries of small-molecule drugs will facilitate the development of new drugs against various diseases, cancers, and HIV.