Ratiometric Fluorescent Biosensor Research Articles

α-Synuclein amplifies cytoplasmic peroxide flux and oxidative stress provoked by mitochondrial inhibitors in CNS dopaminergic neurons in vivo

Convergent evidence implicates impaired mitochondrial function and α-Synuclein accumulation as critical upstream events in the pathogenesis of Parkinson's disease, but comparatively little is known about how these factors interact to provoke neurodegeneration. We previously showed that α-Synuclein knockdown protected rat substantia nigra dopaminergic neurons from systemic exposure to the mitochondrial complex I inhibitor rotenone. Here we show that motor abnormalities prior to neuronal loss in this model are associated with extensive α-Synuclein-dependent cellular thiol oxidation. In order to elucidate the underlying events in vivo we constructed novel transgenic zebrafish that co-express, in dopaminergic neurons: (i) human α-Synuclein at levels insufficient to provoke neurodegeneration or neurobehavioral abnormalities; and (ii) genetically-encoded ratiometric fluorescent biosensors to detect cytoplasmic peroxide flux and glutathione oxidation. Live intravital imaging of the intact zebrafish CNS at cellular resolution showed unequivocally that α-Synuclein amplified dynamic cytoplasmic peroxide flux in dopaminergic neurons following exposure to the mitochondrial complex I inhibitors MPP+ or rotenone. This effect was robust and clearly evident following either acute or prolonged exposure to each inhibitor. In addition, disturbance of the resting glutathione redox potential following exogenous hydrogen peroxide challenge was augmented by α-Synuclein. Together these data show that α-Synuclein is a critical determinant of the redox consequences of mitochondrial dysfunction in dopaminergic neurons. The findings are important because the mechanisms underlying α-Synuclein-dependent reactive oxygen species fluxes and antioxidant suppression might provide a pharmacological target in Parkinson's disease to prevent progression from mitochondrial dysfunction and oxidative stress to cell death.

Development of a Ratiometric Fluorescent Glucose Sensor Using an Oxygen-Sensing Membrane Immobilized with Glucose Oxidase for the Detection of Glucose in Tears.

Glucose concentration is an important parameter in biomedicine since glucose is involved in many metabolic pathways in organisms. Many methods for glucose detection have been developed for use in various applications, particularly in the field of healthcare in diabetics. In this study, ratiometric fluorescent glucose-sensing membranes were fabricated based on the oxygen levels consumed in the glucose oxidation reaction under the catalysis of glucose oxidase (GOD). The oxygen concentration was measured through the fluorescence quenching effect of an oxygen-sensitive fluorescent dye like platinum meso-tetra (pentafluorophenyl) porphyrin (PtP) by oxygen molecules. Coumarin 6 (C6) was used as a reference dye in the ratiometric fluorescence measurements. The glucose-sensing membrane consisted of two layers: The first layer was the oxygen-sensing membrane containing polystyrene particles (PS) doped with PtP and C6 (e.g., PS@C6^PtP) in a sol–gel matrix of aminopropyltrimethoxysilane and glycidoxypropyltrimethoxysilane (GA). The second layer was made by immobilizing GOD onto one of three supporting polymers over the first layer. These glucose-sensing membranes were characterized in terms of their response, reversibility, interferences, and stability. They showed a wide detection range to glucose concentration in the range of 0.1 to 10 mM, but high sensitivity with a linear detection range of 0.1 to 2 mM glucose. This stable and sensitive ratiometric fluorescent glucose biosensor provides a reliable way to determine low glucose concentrations in blood serum by measuring tear glucose.

Ratiometric fluorescent biosensor for microRNAs imaging in living cells

Two-dimensional nanomaterials have been widely used as fluorescent quenching platforms and nanocarriers of fluorophore-labelled nucleic acid probes to design nanoprobes for fluorescence imaging of intracellular microRNAs (miRNAs). However, the detection result was simply dependent on single fluorescent signal of fluorophore-labelled nucleic acid probes. Herein, a ratiometric fluorescent biosensor was proposed by utilizing protonated phenyl-doped carbon nitride (PDCN) nanosheets not only as a fluorescent quenching platform and nanocarrier of ROX-ssDNA probes but also as an internal fluorescent reference. After specific recognition and hybridization of target miRNAs, the fluorescence of ROX-ssDNA probes was recovered while the fluorescence of protonated PDCN nanosheets remained unchanged. By using the fluorescence ratio of ROX-ssDNA probes and protonated PDCN nanosheets as the response signal, this biosensor was used to detect DNA analog of miRNA-224 over a linear range of 1– 50 nmol L−1 with a limit of detection 0.2 nmol L−1. Finally, this ratiometric fluorescent biosensor was applied for fluorescence imaging of miRNA-224 in HeLa cells and HepG2 cells, which indicated this biosensor possessed a great potential for quantitative detection of miRNAs by fluorescence imaging in living cells.

A ratiometric fluorescent biosensor based on conjugated polymers for sensitive detection of nitroreductase and hypoxia diagnosis in tumor cells

The over-expressed nitroreductase (NTR) is a common biomarker of tumor hypoxia. Thus, achieving sensitive and accurate detection of NTR is significant for the guidance of tumor therapy. In this work, a novel ratiometric fluorescent biosensor based on water-soluble conjugated polymers PFP-NA has been established for NTR sensing and hypoxia diagnosis. The 1,8-naphthalimide (NA) modified with p-nitrobenzene is linked to the side chain of PFP through covalent bonds, which leads to the simultaneous occurrence of fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET). Due to the fluorescence of NA being quenched by PET, the emission ratio of NA to PFP backbone is quite low (around 0.09). When NTR exists, the nitro group is reduced to an amino group, leading to PET being blocked. Thus the ratio increases greatly (>54-fold), bringing the calculated detection limit as low as 19.7 ng/mL. Importantly, the fluorescence of PFP backbone remains unchanged, which enables it being used as an internal reference. Moreover, our ratiometric probe exhibits high selectivity and low cytotoxicity, and can achieve intracellular detection of NTR and hypoxia diagnosis in tumor cells. Briefly, this work provides a promising ratiometric biosensor for sensitive and accurate nitroreductase detection and hypoxia imaging.

Switch-conversional ratiometric fluorescence biosensor for miRNA detection

MicroRNAs play a crucial role in regulating gene expression and cellular function. Reliable detection of miRNA is highly demanded in clinical diagnosis and therapy. Herein, we designed a structure-convertible DNA switch and constructed a novel switch-conversional ratiometric fluorescence biosensor (SCRF biosensor) for highly sensitive miRNA detection by the use of amplicon fragments to convert the structure of the switch. The DNA switch was a sophisticated designed single-strand DNA with a stem-loop structure and modified with two fluorophores (Cy3 and Cy5) and one quencher at specific sites of the switch. Amplicon fragments (c*) were produced by an exponential amplification reaction. When the c* hybridized to the loop of a DNA switch, the structure of the switch would convert, and fluorescence resonance energy transfer occurred between Cy5 and Cy3. Then two fluorescence signals with different trends would be observed. As a result, by the ratio of the two signals, we can quantitatively and quickly detect the target miRNA with the concentration range from 100 fM to 100 nM and the excellent detection limit down to 70.9 fM, providing this new SCRF biosensor broad application prospects.

Solvothermal and Ultrasonic Preparation of Two Unique Cluster-Based Lu and Y Coordination Materials: Metal-Organic Framework-Based Ratiometric Fluorescent Biosensor for an Ornidazole and Ronidazole and Sensing Platform for a Biomarker of Amoeba Liver Abscess.

Through powerful solvothermal and facile ultrasonic synthetic strategies, two unique cluster-based lanthanide Lu and Y nanoporous metal organic frameworks (MOFs) have been successfully prepared, namely, {[Lu2(L)2]·2DMF·H2O}n (Lu-MOF) and [Y(L)(DMF)0.75]n (Y-MOF) (H3L = terphenyl-3,4'',5-tricarboxylic acid). In addition, both the morphologies and nanosizes of Lu-MOF and Y-MOF materials also have been deliberately tuned by adjustable ultrasonic conditions including irradiation time (40, 60, and 80 min) and power (70 w, 100 w). Currently, it is noted that the abuse of antibiotics such as ornidazole and ronidazole leads to great damage to human health, and therefore the development of highly effective and facile detection methods for ornidazole and ronidazole is quite important. Herein, to improve the fluorescent sensing sensitivity of antibiotics, Eu3+ and Tb3+ have been introduced into Lu-MOF (under a solvothermal preparation method) to fabricate a dual-emission hybrid material Eu3+/Tb3+@Lu-MOF through a postsynthesis strategy, which can be successfully applied as a self-calibrated ratiometric fluorescent sensor for ornidazole and ronidazole with high selectivity and sensitivity (the Ksv value for ornidazole is 1.0854 × 106 [M-1], and the Ksv value for ronidazole is 1.0595 × 107 [M-1]) and low detection limit values (2.85 nM for ornidazole and 26.7 nM for ronidazole). On the other hand, amoeba liver abscess (ALA) will easily lead to irregular fever, night sweats, and other tortured symptoms; C-reactive protein autoantibody (CRP Ab) is the important biomarker for the detection of ALA. Given this, Y-MOF (under the solvothermal preparation method) also has been successfully designed to combine FAM-labeled NH-ssDNA to construct the scarcely reported excellent hybrid FAM-labeled NH-ssDNA/Y-MOF sensing platform for the highly effective discrimination of CRP Ab with excellent sensitivity and selectivity in real samples such as human serum solution.

Assembly of black phosphorus quantum dots-doped MOF and silver nanoclusters as a versatile enzyme-catalyzed biosensor for solution, flexible substrate and latent fingerprint visual detection of baicalin

In this work, a versatile enzyme-catalyzed biosensor was developed by using the assembled nanohybrids of black phosphorus quantum dots (BPQDs)-doped metal-organic frameworks (MOF) and silver nanoclusters (AgNCs). The nanohybrids of AgNCs/BPQDs/MOF exhibit dual-emissive fluorescence (FL) centers at 630 nm (red) and 535 nm (blue) under excitation at 440 nm. Baicalin enhances the activity of catalase and catalytic decomposition of H2O2. With increase of baicalin contents in the mixture containing nanohybrids, catalase and H2O2, the catalase-caused decomposition of H2O2 was accelerated and the excessive H2O2 was consumed. Baicalin can restrain the oxidation capability of H2O2. The red-FL (response signal) of AgNCs adhering to MOF increases, while blue-FL (reference signal) of BPQDs doped into MOF has negligible changes. A new ratiometric FL biosensor was designed based on nanohybrids and enzyme-catalyzed reaction. This biosensor enables the detection of baicalin in the range of 0.01–500 μg mL−1, with a limit of detection of 3 ng mL−1. This biosensor has high sensitivity, selectivity and stability for baicalin detection in practical samples. Especially, it performed the solution, flexible substrate and latent fingerprint visual detection of baicalin through direct observation of FL color shades with naked eyes. This work explored a facile and efficient semi-quantitative method for versatile FL visual detection, which can promote the development of advanced chemo/bio-sensors and analysis methods.

PiQSARS: A pipeline for quantitative and statistical analyses of ratiometric fluorescent biosensors

Genetically encoded ratiometric fluorescent probes are cutting-edge tools in biology. They allow precise and dynamic measurement of various physiological parameters within cell compartments. Because data extraction and analysis are time consuming and may lead to inconsistencies between results, we describe here a standardized pipeline for•Semi-automated treatment of time-lapse fluorescence microscopy images.•Quantification of individual cell signal.•Statistical analysis of the data.First, a dedicated macro was developed using the FIJI software to reproducibly quantify the fluorescence ratio as a function of time. Raw data are then exported and analyzed using R and MATLAB softwares. Calculation and statistical analysis of selected graphic parameters are performed. In addition, a functional principal component analysis allows summarizing the dataset. Finally, a principal component analysis is performed to check consistency and final analysis is presented as a visual diagram. The method is adapted to any ratiometric fluorescent probe. As an example, the analysis of the cytoplasmic HyPer probe in response to an acute cell treatment with increasing amounts of hydrogen peroxide is shown. In conclusion, the pipeline allows to save time and analyze a larger amount of samples while reducing manual interventions and consequently increasing the robustness of the analysis.

A ratiometric fluorescent biosensor for the sensitive determination of α-glucosidase activity and acarbose based on N-doped carbon dots.

In this work, a novel ratiometric fluorescent platform for α-glucosidase (α-glu) and its inhibitor was constructed based on N-doped carbon dots (N-CDs). The α-glucosidase present can catalyze the release of hydroquinone (HQ) from α-arbutin. Then, the generated HQ can be oxidized and copolymerized with polyethyleneimine (PEI) to form a yellowish green fluorescence copolymer (PHQ-PEI) with intense fluorescence emission at 510 nm. When the PHQ-PEI was formed, blue fluorescence of N-CDs at 425 nm was decreased, whereas the fluorescence of PHQ-PEI at 510 nm increased sharply as a result of the fluorescence resonance energy transfer (FRET) effect between N-CDs and PHQ-PEI. However, in the presence of acarbose, the activity of α-glucosidase is inhibited, and α-arbutin cannot be hydrolyzed to hydroquinone, leading to the fluorescence recovery of N-CDs at 425 nm and the fluorescence decrease of PHQ-PEI at 510 nm. The linear range from 0.2 to 1.6 mU mL-1 and 25-150 μmol L-1 was obtained for α-glucosidase and acarbose detection, respectively, and the detection limit (LOD) for α-glucosidase and acarbose was as low as 0.082 mU mL-1 and 14.5 μmol L-1. Thus, a ratiometric fluorescent sensor with good sensitivity and high specificity was established for α-glucosidase assay and satisfactory results were acquired in real sample determination.

Ratiometric fluorescence method for ctDNA analysis based on the construction of a DNA four-way junction.

We present a novel ratiometric fluorescent biosensor for ctDNA analysis based on the construction of a DNA four-way junction (FWJ). Three fuel strands for the FWJ are firstly designed and prepared. Another essential strand for the formation of the structure is the DNA product generated from target ctDNA initiated strand displacement amplification. With the transformation of the DNA structure, the FRET states of two fluorophores change and the ratiometric fluorescence response can be recorded to indicate the level of the initial ctDNA. The proposed method also has excellent capability to discriminate mismatches and shows potential practical utility for clinical samples.

A zirconium-porphyrin MOF-based ratiometric fluorescent biosensor for rapid and ultrasensitive detection of chloramphenicol

An ultrasensitive and rapid detection of trace antibiotics is imperative for food safety and public health. Herein, we present a ratiometric fluorescent sensing strategy based on an aptamer labeled with a fluorescent dye and a highly stable zirconium-porphyrin MOF (PCN-222) as a fluorescence quencher for the high-efficiency detection of chloramphenicol (CAP). PCN-222 exhibits a strong adsorption ability toward the dye-labeled aptamer through π-π stacking, electrostatic, hydrogen bond, and coordination interactions. Experimental and simulation studies confirm that PCN-222 demonstrates a high quenching efficiency via fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) processes. In the presence of CAP, dye-labeled aptamers are released from the PCN-222 surface, resulting in the recovery of fluorescence. This proposed biosensor allows the complete detection of CAP within 26 min. For ratiometric measurement, its detection limit is as low as 0.08 pg mL−1 with a wide detection range from 0.1 pg mL−1 to 10 ng mL−1. It is successfully applied to analyze CAP in milk and shrimp samples, and its results are consistent with those of the commercial ELISA kit. This biosensor not only enables the rapid, ultrasensitive, and highly specific detection of CAP but also reveals excellent universality and multiplexed analysis performance.

Ratiometric fluorescence sensor for organophosphorus pesticide detection based on opposite responses of two fluorescence reagents to MnO2 nanosheets

The detection of organophosphorus pesticides (OPs) has received considerable attention for their great harm to human beings. Herein, a novel ratiometric fluorescence biosensor was constructed for the determination of OPs by using Scopoletin (SC) and Amplex Red (AR) as probe pairs that have opposite responses to MnO2 nanosheets (MnO2 NS). MnO2 NS possess peroxidase-like catalytic activity, which could quench the fluorescence of SC as well as enhance the fluorescence of the non-fluorescent substance AR by oxidation. In the absence of OPs, acetylcholinesterase (AChE) hydrolyzed acetylcholine chloride (ATCh) into choline (TCh) and acetate. TCh led the decomposition of MnO2 NS to manganese ions (Mn2+), increasing signal of SC and decreasing signal of AR. In the presence of OPs, the activity of AChE was inhibited and the decomposition of MnO2 NS was hindered, therefore the fluorescence intensity of SC was weak and the fluorescence intensity of AR had an obvious increase. Moreover, under the optimal conditions, the ratio of fluorescence intensity response recorded on the AR/SC increases with increasing the concentration of DDVP. The method has wider linear range of 5.0 pg/mL ∼500 ng/mL with a detection limit of 1.6 pg/mL, which is superior to previously reported methods. This strategy has also been applied to a visual observation based on the color change of the solution under UV light.

Chameleon silver nanoclusters for ratiometric sensing of miRNA

Chameleon silver nanoclusters (AgNCs) have attracted substantial attention over traditional AgNCs, which provide new possibilities for the construction of ratiometric fluorescent biosensors. Herein, a novel method for sensitive and selective detection of miRNA is developed coupling chameleon AgNCs with hybridization chain reaction. Generally, the signal DNA probe is designed to contain two pieces of DNA templates for the synthesis of AgNCs, which are in close proximity due to the hairpin structure. A series of hybridization reactions can be triggered by target miRNA, which open the hairpin of signal probe and separate the two pieces of segmental AgNCs. Controllable color switching from red to yellow is thus achieved. Note that signal generation and amplification are integrated into one step, which is a great advance compared with previous amplified systems. The proposed strategy provides a sensitive fluorescent detection of miRNA down to 2.8 pM with high selectivity. Therefore, this work not only brings novel insights into fluorescence regulation of DNA-templated AgNCs, but also holds great potential for the application of miRNA diagnostics.

Rapid synthesis of Au/Ag bimetallic nanoclusters with highly biochemical stability and its applications for temperature and ratiometric pH sensing

Herein, we developed a simple and rapid strategy to synthesize gold/silver bimetallic nanoclusters (Au/Ag NCs) with highly biochemical stability by a one-pot route. The Au/Ag NCs were obtained via a chemical reduction procedure in alkaline aqueous solution at 75 °C within only 20 min by employing bovine serum albumin (BSA) as both ligand and reductant. The as-obtained Au/Ag NCs displayed bright orange fluorescence with an emission peak located at 570 nm and temperature-dependent fluorescence property, which were utilized as fluorescent thermometer directly. More intriguingly, the Au/Ag NCs were very stable against various pH values, ions, biothiols, H2O2, fetal bovine serum (FBS), RPMI 1640 medium and amino acids. Taking advantage of the excellent biochemical stability, a ratiometric fluorescence biosensor, fluorescein-5-isothiocyanate (FITC)-Au/Ag NCs, was constructed for pH sensing based on the incorporation of FITC into the Au/Ag NCs. Furthermore, the ratiometric pH sensor was also successfully applied on the model of HeLa cells.

MnO2 nanosheet-mediated ratiometric fluorescence biosensor for MicroRNA detection and imaging in living cells

MicroRNA (miRNA) plays significant roles in cell proliferation, differentiation and apoptosis, and has been considered to be valuable biomarker for cancer. Accurate and sensitive detection of miRNA is crucially significant for cancer diagnosis and treatment. Here, a MnO2 nanosheet-mediated ratiometric fluorescence biosensor was designed for miRNA detection and imaging in living cells. It contained MnO2 nanosheets acting as DNA carrier, and fluorescent donor (FAM)-labeled hairpin H1 (recognition probe) and fluorescent acceptor (TAMRA)-labeled hairpin H2 (amplification probe). When the biosensor entered cell by endocytosis, MnO2 nanosheets were degraded to Mn2+ via intracellular glutathione (GSH) and the adsorbed hairpins H1 and H2 were released. The intracellular target miRNA-21 hybridized with the recognition unit of H1 to initiate catalyzed hairpin assembly (CHA) and a large amount of H1-H2 duplexes were produced. This brought fluorescent donor FAM and fluorescent acceptor TAMRA into close proximity to produce fluorescence resonance energy transfer (FRET), inducing a ratiometric fluorescent response (donor signal decreased and acceptor signal enhanced) for miRNA-21 detection. Furthermore, this method could be applied to differentiate the expression levels of miRNA-21 in HeLa, HepG-2 and L02 cells. These results indicated that the proposed method possessed great potential in the early diagnosis of miRNA-related diseases.

Hydrogen Peroxide-Assisted Ultrasonic Synthesis of BCNO QDs for Anthrax Biomarker Detection.

A facile 3% hydrogen peroxide-assisted ultrasonic synthetic strategy is demonstrated to successfully synthesize fluorescent boron carbon oxynitride quantum dots (BCNO QDs). The obtained BCNO QDs exhibit intense blue fluorescence and favorable biocompatibility and water solubility. The quantum yield of the BCNO QDs is 19.9%. Owing to the absorbance energy-transfer emission effect, an efficient ratiometric fluorescence biosensor is developed for anthrax biomarker detection based on the BCNO QD-ethylenediaminetetraacetic acid disodium salt-Eu3+ complex. Under optimal conditions, the detection limit of the anthrax biomarker is 0.5 nM. Furthermore, the sensitivity of the system was evaluated by Bacillus subtilis spores and with the detection limit as low as 1.95 × 106 spores. On combining a smartphone with the home-made BCNO QD test paper, the lowest recorded visual detection limit of 1.0 μM anthrax biomarker was achieved using a portable UV lamp. The fast response speed, excellent sensitivity, and selectivity of the approach show potential applications in clinical analysis.

A split aptamer-labeled ratiometric fluorescent biosensor for specific detection of adenosine in human urine.

A dual-emission ratiometric fluorometric aptasensor is presented for highly specific detection of adenosine. An adenosine binding aptamer (ABA) was split into two halves (termed as ABA1 and ABA2). ABA1 was covalently bound to blue-emitting carbon dots (with excitation/emission maxima at 365/440nm) as responsive fluorophore (referred to as ABA1-CDs). ABA2 was linked to red-emitting silica-coated CdTe quantum dots (with excitation/emission maxima at 365/613nm) acting as internal reference and referred to as ABA2-QDs@SiO2. Upon addition of graphene oxide, the fluorescence of ABA1-CDs is quenched. After subsequent addition of ABA2-QDs@SiO2 and different amounts of adenosine, the blue fluorescence is recovered and causes a color change from red to royalblue. The method represents a ratiometric turn-on assay for visual, colorimetric and fluorometric determination of adenosine. The limit of detection is as low as 2.4nM in case of ratiometric fluorometry. The method was successfully applied to the determination of adenosine in (spiked) human urine. Recoveries range from 98.8% to 102%. Graphical abstract Adenosine binding aptamer1-carbon dots (ABA1-CDs) can absorb on graphene oxide (GO) via π stacking. This causes fluorescence to be quenched by fluorescence resonance energy transfer (FRET). After addition of ABA2-silica-coated quantum dots (ABA2-QDs@SiO2) and adenosine, binding of adenosine to two pieces of aptamers forms acomplex (ABA1-CD/adenosine/ABA2-QD@SiO2)which dissociates from GO. As a result, fluorescence is recovered.

A reusable ratiometric fluorescent biosensor with simple operation for cysteine detection in biological sample

Highly sensitive detection of cysteine is critical for early warning and diagnosis of cysteine-associated diseases. For the first time, herein, a reusable and ratiometric fluorescent biosensor with simple operation was developed for sensitive and rapid detection of Cys on the basis of configuration transformation of guanine-rich DNA (G-DNA). The sensing system is quite simple and consisted of G-DNA and Ag+ with NMM (N-methylmesoporphyrin IX) and terbium ion (Tb3+) as the two fluorescence probes. The Ag+ assists the G-DNA to keep its random single-strand structure (ssG-DNA), generating a distinct fluorescence of Tb3+ and a faint fluorescence of NMM. The analyte of cysteine (Cys) can strongly coordinate with Ag+, thus, the ssG-DNA is transferred into G-quadruplex (G4) configuration. The fluorescence of NMM is then outstandingly augmented, while the fluorescence of Tb3+ is significantly depressed. With the fluorescent intensity ratio of FNMM/FTb as readout signal, the detection limit of the developed ratiometric fluorescent biosensor reaches as low as 4.1 nM, confirming the excellent sensitivity. After completing the Cys detection, Ag+ is further added to disrupt the G-quadruplex into random single-strand G-DNA, which restores the sensing system into the initial state. The reversible configuration change between G-quadruplex and single-stand G-DNA endows the as-developed sensing system with charming reusability for specific detection of Cys. The developed sensing assay is cost-effective and operated in an enzyme-free and label-free condition, providing a straightforward and effective “mix-then-test” strategy to meet the requirement for practical application in disease monitoring and diagnosis.

Ratiometric fluorescence biosensor based on DNA/miRNA duplex@CdTe QDs and oxidized luminol as a fluorophore for miRNA detection

Breast cancer is the second most diagnosed cancer in women worldwide. Hence, the detection of prognostic and diagnostic biomarkers can improve the patient's quality of life during the course of illness and treatment. Most recently, microRNAs (miRNAs) have been widely studied for non-invasive prediction of prognosis markers in the diagnosis of breast cancer. Herein, we reported a ratiometric fluorescence nanobiosensor for detection of miR-155. The biosensor comprises of 3-mercapropionic acid-coated cadmium telluride (CdTe) quantum dots and oxidized luminol (Lumox) exhibiting emission peaks at 550 and 440 nm, respectively, under single-wavelength excitation (350 nm). In the presence of miR-155, CdTe QDs were aggregated and their fluorescence was quenched via interacting strongly at their metal centers, Cd, with heteroduplex formed between the DNA probe and miR-155. Then, by addition of blue-emitting Lumox with their constant fluorescence emission, a ratiometric means of miR-155 detection was developed. The relative fluorescence intensity ration is directly proportional to the concentration of miR-155 between 20.0 and 100.0 pM. The detection limit is 12.0 pM. This novel assay is “light-on” and has been successfully applied for the detection of miRNA in MCF-7 and HEK 293 cell lysates as real samples.

A ratiometric fluorescent biosensor based on cascaded amplification strategy for ultrasensitive detection of kanamycin

Antibiotic residues in food products significantly threaten public health. In the present work, a fluorescence resonance energy transfer (FRET)-based ratiometric fluorescent biosensor was developed for high sensitive and selective detection of kanamycin by combining aptamer-recognition assay and signal amplification strategy. To improve detection sensitivity, the optimum FRET efficiency of donor-acceptor was explored by modulating the separation distance between the donor (Cy3) and the acceptor (Cy5). In the developed biosensing system, kanamycin could be trapped by its aptamer, which triggered a polymerase-catalyzed amplification (PCA) process to autonomously generate a single-strand DNA. In subsequence, the generated DNA strand initiated the second amplification process of catalyzed hairpin assembly (CHA) to cause fluorescence decreasing of Cy3 and increasing of Cy5. The signal change caused by FRET of donor (Cy3)-acceptor (Cy5) was then augmented by the PCA and CHA processes. The constructed biosensors results in high sensitive detection of kanamycin as low as 0.29 nM within a linear range from 1.0 nM to 80.0 nM. The potential application of the developed biosensing system was evaluated with milk as real sample. The provided strategy could serve as a prototype for developing FRET-based ratiometric bioassays.