On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1.     

A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13 fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3 min.     

Ratiometric fluorescence transduction by hybridization after isothermal amplification for determination of zeptomole quantities of oligonucleotide biomarkers with a paper-based platform and camera-based detection

Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)–probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ∼100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non-ratiometric QD-FRET transduction method. The selectivity of the hybridization assays was demonstrated by the detection of single nucleotide polymorphism.     

Quantum dots as donors in fluorescence resonance energy transfer for the bioanalysis of nucleic acids,proteins, and other biological molecules

Quantum dots (QDs) have a number of unique optical properties that are advantageous in the development of bioanalyses based on fluorescence resonance energy transfer (FRET). Researchers have used QDs as energy donors in FRET schemes for the analysis of nucleic acids, proteins, proteases, haptens, and other small molecules. This paper reviews these applications of QDs. Existing FRET technologies can potentially be improved by using QDs as energy donors instead of conventional fluorophores. Superior brightness, resistance to photobleaching, greater optimization of FRET efficiency, and/or simplified multiplexing are possible with QD donors. The applicability of the Förster formalism to QDs and the feasibility of using QDs as energy acceptors are also reviewed.

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Figure A ligand capped core/shell quantum dot acting as energy donor in a FRET process with aconjugated Cy3 labeled oligonucleotide

     

基于锰掺杂量子点的噁喹酸荧光共振能量转移检测研究

基于噁喹酸对锰掺杂硫化锌量子点的荧光猝灭作用,建立了一种噁喹酸荧光共振能量转移检测方法.噁喹酸对量子点的荧光猝灭是由于生成了新的复合物而造成的静态猝灭,二者相互作用过程中焓变ΔH ＜ 0,熵变ΔS ＜ 0,分子间作用力为氢键或范德华力.在0～65 μg/L线性范围内,噁喹酸质量浓度与量子点荧光抑制率呈现良好的线性关系(...     

Fungal pathogenic nucleic acid detection achieved with a microfluidic microarray device

Detection of polymerase chain reaction (PCR) products obtained from cultured greenhouse fungal pathogens, Botrytis cinerea and Didymella bryoniae has been achieved using a previously developed microfluidic microarray assembly (MMA) device. The flexible probe construction and rapid DNA detection resulted from the use of centrifugal pumping in the steps of probe introduction and sample delivery, respectively. The line arrays of the oligonucleotide probes were “printed” on a CD-like glass chip using a polydimethylsiloxane (PDMS) polymer plate with radial microfluidic channels, and the sample hybridizations were conducted within the spiral channels on the second plate. The experimental conditions of probe immobilization and sample hybridization were optimized, and both complementary oligonucleotides and PCR products were tested. We were able to achieve adequate fluorescent signals with a sample load as small as 0.5 nM (1 μL) for oligonucleotide samples; for PCR products, we achieved detection at the level of 3 ng.     

Fluorescence-based nucleic acid detection and microarrays

Recent analytical innovations for nucleic acid detection have revolutionized the biological sciences. Single nucleic acid sequence detection methods have been expanded to incorporate multiplexed detection strategies. A variety of nucleic acid detection formats are now available that can address high throughput genomic interrogation. Many of these parallel detection platforms or arrays, employ fluorescence as the signaling method. Fluorescence-based assays offer many advantages, including increased sensitivity, safety and multiplexing capabilities, as well as the ability to measure multiple fluorescence properties. Multiplexed microarray platforms provide parallel detection capabilities capable of measuring thousands of simultaneous responses. This review will discuss both single target detection and microarray applications with a focus on gene expression and pathogenic microorganism (PM) detection.     

Lateral flow devices for nucleic acid analysis exploiting quantum dots as reporters

There is a growing interest in the development of biosensors in the form of simple lateral flow devices that enable visual detection of nucleic acid sequences while eliminating several steps required for pipetting, incubation and washing out the excess of reactants. In this work, we present the first dipstick-type nucleic acid biosensors based on quantum dots (QDs) as reporters. The biosensors enable sequence confirmation of the target DNA by hybridization and simple visual detection of the emitted fluorescence under a UV lamp. The ‘diagnostic’ membrane of the biosensor contains a test zone (TZ) and a control zone (CZ). The CZ always fluoresces in order to confirm the proper function of the biosensor. Fluorescence is emitted from the TZ, only when the specific nucleic acid sequence is present. We have developed two general types of QD-based nucleic acid biosensors, namely, Type I and Type II, in which the TZ consists of either immobilized streptavidin (Type I) or immobilized oligodeoxynucleotides (Type II). The control zone consists of immobilized biotinylated albumin. No purification steps are required prior to the application of the DNA sample on the strip. The QD-based nucleic acid biosensors performed accurately and reproducibly when applied to (a) the visual detection of PCR amplification products and (b) visual genotyping of single nucleotide polymorphisms (SNPs) in human genomic DNA from clinical samples. As low as 1.5 fmol of double-stranded DNA were clearly detected by naked eye and the dynamic range extended to 200 fmol. The %CV were estimated to be 4.3–8.2.     

Buffer enhanced bioluminescence resonance energy transfer sensor based on Gaussia luciferase for in vitro detection of protease

Bioluminescence resonance energy transfer (BRET) has gained favors in recent years as a detection technology for protease activity due to its extreme reliability, high sensitivity and low intrinsic backgrounds. Because of the sensitivity of the donors, substrates and the acceptors, it is expected that BRET systems are sensitive to buffer environments. However, no systematic study has been reported on how buffer components would affect the BRET ratio, and thus affect the determination of protease activity based on BRET. We present here that several environmental factors, including buffer agents, pH and divalent metal ions, influenced BRET ratio significantly, when humanized Gaussia luciferase (hGluc) was utilized as the donor and enhanced yellow fluorescence protein (EYFP) as the acceptor. Based on these findings, an enhancing solution was optimized to improve the performance of the BRET sensor for analysis of enterokinase activity in vitro, resulting in 10-fold and 7-fold improvement of the sensitivity and the detection limit, respectively. We anticipate the system will be applicable for improving performance of other in vitro BRET protease sensors, especially when the optimal conditions for protease activity would severely affect the BRET signal.     

Resonance energy transfer methods of RNA detection

Quantitation of RNA is important in diagnostics, environmental science, and basic biomedical research. RNA is considered a signature for pathogen identification, and its expression profile is linked with disease pathogenesis, allowing for biomarker identification. RNA-based diagnostics is an emerging field of research. This expansion of interest in studying RNA has generated demand for its accurate and sensitive detection. Several methods have therefore been developed to detect RNA. Resonance energy transfer methods of RNA detection are highly promising in terms of simplicity and high sensitivity. In this review, we have focused on the latest developments in resonance energy transfer methods of RNA detection that utilize various probe designs. The probe designs discussed here are molecular beacons, quenched autoligation probes, and linear oligonucleotide probes. Resonance energy transfer methods based on both fluorescence and bioluminescence detection are discussed.     

RNA detection using peptide-inserted <Emphasis Type="Italic">Renilla</Emphasis> luciferase

A novel complementation system with short peptide-inserted-Renilla luciferase (PI-Rluc) and split-RNA probes was constructed for noninvasive RNA detection. The RNA binding peptides HIV-1 Rev and BIV Tat were used as inserted peptides. They display induced fit conformational changes upon binding to specific RNAs and trigger complementation or discomplementation of Rluc. Split-RNA probes were designed to reform the peptide binding site upon hybridization with arbitrarily selected target RNA. This set of recombinant protein and split-RNA probes enabled a high degree of sensitivity in RNA detection. In this study, we show that the Rluc system is comparable to Fluc, but that its detection limit for arbitrarily selected RNA (at least 100 pM) exceeds that of Fluc by approximately two orders of magnitude.     

Capillary waveguide nucleic acid based biosensor

Micro-capillaries are finding increasing utility in the development of portable analytical sensors. We present design guidelines for optimizing the collection of free propagating fluorescence for capillary waveguide sensors used in the detection of nucleic acids. A dual function integrated opto/fluid connector is also described. Evanescent wave excitation of the coating layer containing a DNA probe is achieved by using a fiber optic ring arrangement for coupling light directly into the capillary wall. The central part of the connector is used for injecting a DNA or RNA target into the capillary channel. In situ hybridization has been used to detect target molecules at a concentration of 30 pg ml⁻¹. The sensor can be regenerated for repeated detection of DNA or RNA targets.     

Study on DNA damage induced by CdSe quantum dots using nucleic acid molecular “light switches” as probe

The calf thymus DNA (ctDNA) damage induced by water-soluble CdSe quantum dots (QDs) was investigated using nucleic acid molecular “light switches” as probe. It was found that little ctDNA was damaged by CdSe QDs without UV irradiation. However, under UV irradiation, ctDNA was nicked by CdSe QDs very clearly. The mechanism of ctDNA damage was also discussed. The results strongly suggested that the ctDNA damage caused by CdSe QDs was not due to photo-induced liberation of Cd²⁺, but due to the production of free radicals and reactive oxygen species.     

Influence of quantum dot's quantum yield to chemiluminescent resonance energy transfer

The resonance energy transfer between chemiluminescence donor (luminol-H₂O₂ system) and quantum dots (QDs, emission at 593 nm) acceptors (CRET) was investigated. The resonance energy transfer efficiencies were compared while the oil soluble QDs, water soluble QDs (modified with thioglycolate) and QD-HRP conjugates were used as acceptor. The fluorescence of QD can be observed in the three cases, indicating that the CRET occurs while QD acceptor in different status was used. The highest CRET efficiency (10.7%) was obtained in the case of oil soluble QDs, and the lowest CRET efficiency (2.7%) was observed in the QD-HRP conjugates case. This result is coincident with the quantum yields of the acceptors (18.3% and 0.4%). The same result was observed in another similar set of experiment, in which the amphiphilic polymer modified QDs (emission at 675 nm) were used. It suggests that the quantum yield of the QD in different status is the crucial factor to the CRET efficiency. Furthermore, the multiplexed CRET between luminol donor and three different sizes QD acceptors was observed simultaneously. This work will offer useful support for improving the CRET studies based on quantum dots.     

Direct Detection of Hemi-methylated DNA by SRA-fused Luciferase Based on Bioluminescence Resonance Energy Transfer

Hemi-methylated DNA is double stranded (ds)DNA where only one of the complementary strands is methylated. Hemi-methylated DNA levels on a tandem repeat NBL2 and Sat2 are increased in ovarian carcinomas. To quantify hemi-methylation levels, a bisulfite-based method is widely used, but this approach requires multiple steps. Therefore, a simple hemi-methylation assay is required for cancer diagnosis and to elucidate the functions of hemi-methylated DNA. In this study, we aimed to develop a homogeneous assay for the detection of the hemi-methylated DNA level using SET- and RING-associated (SRA) domain-fused luciferase (SRA-luciferase), which recognizes hemi-methylated DNA. In the assay, the hemi-methylation level was quantified by measuring the bioluminescence resonance energy transfer signal between the fusion protein and the DNA intercalating dye. The fusion protein specifically recognizes hemi-methylated DNA to excite the dye by luciferase luminescence. Our results demonstrated that the hemi-methylation level was easily quantified within 35?min with an R² value of 0.99.     

Analysis of dimeric cyanine–nucleic acid dyes by capillary zone electrophoresis in N,N-dimethylacetamide as non-aqueous organic solvent

A method based on capillary zone electrophoresis is presented for the determination of the purity of commercial dimeric cyanine dyes (TOTO, YOYO, BOBO, all -1 and -3 species, LOLO-1, POPO-1) that are common as fluorescent probes for nucleic acid staining. These dyes are tetracharged cations, and have a strong tendency to interact with negatively charged centres, where they are rapidly adsorbed, especially from aqueous solutions. Thus anionic sites at the capillary wall must be avoided, and aqueous buffers are not suitable. The method introduced here avoids both complications, using non-aqueous N,N-dimethylacetamide as solvent, and suppressing the dissociation of silanol groups at the capillary surface due to selection of acidic separation conditions (20 mmol/l perchloric acid as background electrolyte). The present method enables the determination of the purity of all 10 dyes in less than 15 min. The selectivity of the method allows separation of at least five main and differentiating a number of unresolved minor contaminants as demonstrated in detail for TOTO-3 as an example. Quantitation (with 100% normalisation of the peak areas) of nine lots of this dye results in a purity between 33 and 87%.     

Fluorescence resonance energy transfer in doubly-quantum dot labeled IgG system

The mouse immunoglobulin G (mouse IgG) as a kind of bio-molecule was labeled with two different luminescent colloidal semiconductor quantum dots (QDs), green-emitting CdTe quantum dots and red-emitting CdTe quantum dots in this work. As a result of the fluorescence resonance energy transfer (FRET) between the two different sizes nanoparticles with mouse IgG as the binding bridge, a significant enhancement of the emission of the red-emitting CdTe quantum dots and the corresponding quenching of the emission of green-emitting CdTe quantum dots were observed. The relationship between the concentration of the mouse immunoglobulin G and the fluorescence intensity ratio (I_a/I_d) of acceptors and donors was studied also. Under optimal conditions, the calibration graph is linear over the range of 0.1–20.0 mg/L mouse IgG.     

The photophysical studies of a mixture of CdTe quantum dots and negatively charged zinc phthalocyanines

Fluorescence resonance energy transfer (FRET) studies were carried out with quantum dots capped with thioglycolic acid (TGA) and 2-mercaptoethanol (2-ME) and negatively charged phthalocyanines {Zn tetracarboxy (ZnTCPc), Zn octacarboxy (ZnOCPc) and Zn tetrasulfo (ZnTSPc) phthalocyinines} in a 0.1 NaOH:EtOH (50:50) solvent mixture. The best overlap between emission spectra of the donor (QDs) and the absorption spectra of the acceptor (ZnPc derivatives) was observed for TGA capped QDs, very little overlap was obtained for 2-ME QDs. ZnTSPc gave the highest FRET efficiency (0.3), with ZnOCPc and ZnTCPc giving a FRET efficiency of 0.2. The Φ_T values of the MPcs generally decreased in the presence of the QD whereas the triplet lifetimes (τ_T) of the ZnPc derivatives were higher in the presence of QDs.