Ratiometric Fluorescent Biosensor Research Articles

Ratiometric fluorescent biosensor for detection and real-time imaging of nitric oxide in mitochondria of living cells

Nitric oxide (NO), a ubiquitous gaseous messenger, plays critical roles in various pathological and physiological progresses. The abnormal levels of NO in organisms are closely related to a large number of maladies. Mitochondria are the main area that produce NO in mammalian cells. Thus, detecting and real-time imaging of NO in mitochondria is of great significance for exploring the biological functions of NO. Herein, a ratiometric fluorescent biosensor (Mito-GNP-pNO520) is developed for sensitive and selective detection and real-time imaging of NO in mitochondria of living cells. The detection is achieved through the fluorescence off-on response of Mito-GNP-pNO520 toward NO. This biosensor shows excellent characteristics, such as high sensitivity toward NO with a low detection limit of 0.25 nM, exclusive selectivity to NO without interference from other substances, good biological stability and low cytotoxicity. More importantly, the biosensor is specifically located in mitochondria, enabling the detection and real-time imaging of endogenous and exogenous NO in mitochondria of living cells. Therefore, our biosensor offers a new approach for dynamic detecting and real-time imaging of NO in subcellular organelles, providing an opportunity to explore new biological effects of NO.

Ratiometric fluorometric assay triggered by alkaline phosphatase: Proof-of-concept toward a split-type biosensing strategy for DNA detection

Herein, a sensitive ratiometric and split-type fluorescent sensing platform has been constructed for DNA detection based on one signal precursor and two fluorescent signal indicators. In this assay, o-phenylenediamine (OPD) was selected as the signal precursor. On one hand, Cu2+ can oxidize OPD to produce 2, 3-diaminophenazine (DAP), which with an emission peak at 555 nm. On the other hand, ascorbic acid (AA) could react with Cu2+ to generate dehydroascorbic acid (DHAA), which could further react with OPD to form 3-(1, 2-dihydroxy ethyl)furo[3, 4-b]quinoxalin-1 (3H)-on (DFQ) with a strong emission peak at 420 nm. As a result, the formation of DAP was inhibited, and leading to the decrease of fluorescence intensity at 555 nm. Alkaline phosphatase (ALP) could catalyze the substrate l-ascorbic acid-2-phosphate (AA2P) to produce AA in situ. Inspired by the successful use of ALP as a biocatalytic marker in bioassay, a split-type ratiometric fluorescent assay has been designed for DNA detection by using H1N1 DNA as the target model. It was realized for ratiometric fluorescent determination of H1N1 in a linear ranging from 50 pM to 1.5 nM with a limit of detection of 10 pM. The novel strategy could reduce the mutual interferences between the biomolecular recognition system and the fluorescence signal conversion system, which improving the accuracy of detection and effectively reducing the background signal. Furthermore, the strategy provided a promising platform for biomarkers detection in the fields of ratiometric fluorescent biosensors and bioanalysis.

An enzyme-free and label-free ratiometric fluorescence signal amplification biosensor based on DNA-silver nanoclusters and catalytic hairpin assembly for tetracycline detection

Sensitive and accurate in antibiotic detection is crucial for food security, environmental conservation, public health, and environmental protection. In this study, we developed an enzyme-free and label-free ratiometric fluorescence biosensor for highly sensitive detection of tetracycline (TET). This biosensor utilized a combination of target-triggered catalytic hairpin assembly (CHA) and DNA-silver nanocluster (DNA-AgNCs) dual-color fluorescent signal probes. Upon TET binding to the aptamer, it initiated the leakage of CHA initiator, resulting in the formation of numerous double-stranded DNA complexes. This process exposed the foothold sequence of hairpin-structured DNA-AgNCs (H3-AgNCs) that exhibit yellow fluorescence. Subsequently, the conformational changes in H3-AgNCs significantly reduced the yellow fluorescence intensity (FI) and reveal the auxiliary DNA-AgNCs (a-DNA-AgNCs) linkage sequence. The binding of a-DNA-AgNCs brought two dark AgNCs close, forming nanocluster dimers emitting red fluorescence. The results demonstrate that the variation in ratiometric signal is linear with TET concentration between 0.1 and 50 ng/mL, with a detection limit as low as 0.086 ng/mL. Additionally, this approach exhibited good recovery results with spiked milk samples. This enzyme-free and label-free combined signal amplification method with proportional signal output based on DNA-AgNCs provides valuable empirical reference for creating low-cost, stable, and highly-sensitive biosensors.

A Ratiometric Fluorescence Biosensor for Detection of Alkaline Phosphatase Via an Advanced Chemometric Model.

In this paper, a ratiometric fluorescence biosensor was introduced for alkaline phosphatase (ALP) detection based on 2-aminopurine (2-Amp) and thioflavin T (ThT)-G-quadruplex system. We designed a special DNA (5'-AGGGTTAGGGTTAGGGTTAGGGAAA/i2-Amp/AAAA-PO4-3', AP) modified with a phosphate moiety at the 3'-end, G-quadruplex at the 5'-end, and a fluorophore (2-Amp) in the middle. In the absence of ALP, the G-rich AP strand could be prone to fold into G-quadruplex structures in the presence of K+. Then, ThT combined with G-quandruplex, resulting in the enhancement of fluorescence emission peak at 485 nm. However, ALP-mediated hydrolysis of the 3'-phosphoryl end promoted the cleavage of AP by the exonuclease I (Exo I), releasing 2-Amp which displayed a strong fluorescence emission peak at 365 nm. Moreover, the quantitative fluorescence model (QFM) was derived for the analysis of the fluorescence measurements obtained by the proposed ratiometric fluorescent biosensor. With the aid of the advanced model, the proposed ratiometric fluorescent biosensor possessed satisfactory results for the detection of ALP in the human serum samples, with accuracy comparable to that of the reference method-the commercial ALP assay kit. Under the optimized experimental conditions, this method exhibited good selectivity and higher sensitivity, and the detection limit was found to be as low as 0.017 U/L. Therefore, it is reasonable to expect that the method had a great potential to detect ALP quantitatively in clinical diagnosis.

A dual-emission ratiometric fluorescent biosensor for ultrasensitive detection of glibenclamide using S-CDs/CdS quantum dots

In the present work, sulfide-doped carbon dots (S-CDs)/cadmium sulfide quantum dots (CdS QDs) ratiometric fluorescent nanosensor has been developed for sensitive and selective determination of glibenclamide (GLC) in biological fluids. The method was based on the quenching effect of GLC on the dual-emission intensity of the S-CDs/CdS QDs system at 420 nm and 650 nm, which are related to S-CDs and CdS QDs, respectively. The fluorimetric data analysis indicated that the fluorescence signals of the system were quenched by adding GLC in a concentration-dependent manner. A good linear relationship was observed between GLC concentration and the quenched fluorescence intensity of the S-CDs/CdS QDs in the range of 0.3 nM−10.0 μM. The limit of detection (LOD) value was estimated to be 0.12 nM. Furthermore, under optimum conditions, GLC was detected in spiked human serum sample (as real media) using the developed ratiometric nanosensor with an accuracy of 99.6%. According to the results, the developed dual-emission system can be used as a reliable method for the quantitative detection of GLC in biological samples.

Real-Time Temperature Sensing Using a Ratiometric Dual Fluorescent Protein Biosensor.

Accurate temperature control within biological and chemical reaction samples and instrument calibration are essential to the diagnostic, pharmaceutical and chemical industries. This is particularly challenging for microlitre-scale reactions typically used in real-time PCR applications and differential scanning fluorometry. Here, we describe the development of a simple, inexpensive ratiometric dual fluorescent protein temperature biosensor (DFPTB). A combination of cycle three green fluorescent protein and a monomeric red fluorescent protein enabled the quantification of relative temperature changes and the identification of temperature discrepancies across a wide temperature range of 4-70 °C. The maximal sensitivity of 6.7% °C-1 and precision of 0.1 °C were achieved in a biologically relevant temperature range of 25-42 °C in standard phosphate-buffered saline conditions at a pH of 7.2. Good temperature sensitivity was achieved in a variety of biological buffers and pH ranging from 4.8 to 9.1. The DFPTB can be used in either purified or mixed bacteria-encapsulated formats, paving the way for in vitro and in vivo applications for topologically precise temperature measurements.

MXene-derived Ti3C2 quantum dots-based ratiometric fluorescence probe for ascorbic acid and acid phosphatase determination

As a burgeoning zero-dimensional nanomaterial, MXene quantum dots which are derived from two-dimensional transition-metal carbides have attracted a lot of interest lately. The transformation of two-dimensional MXene into zero-dimensional structures of a few nanometers enables these recently discovered materials to possess an exceptional quantum size effect and remarkable photoluminescence characteristics. Hence, we synthesized N doped Ti3C2 MXene quantum dots (N-Ti3C2 MQDs) exhibiting bright blue fluorescence through small-molecule amine-assisted solvothermal strategy. In this work, we constructed a novel ratiometric fluorescence biosensor for the determination of ascorbic acid (AA) and acid phosphatase (ACP) based on the excellent optical performance of N-Ti3C2 MQDs and combined o-phenylenediamine (OPD). This ratiometric strategy was established based on the inner filter effect between N-Ti3C2 MQDs and the oxide of OPD. Sensitive fluorometric analyses of AA and ACP were achieved successfully in the respective ranges of 2–240 μM and 0.15–3.75 U/L with respective limits of detection down to 0.82 μM and 0.02 U/L. Moreover, the response time of this fluorescence sensor for AA and ACP determination was less than 1.5 h. The intraday and interday relative standard deviations for AA and ACP assay were all below 8.3 %, suggesting the good reproducibility of this sensor. In addition, the profuse color changed under UV light with analytes concentrations increased and exhibited great potential for visual detection and on-site application. The proposed method was further utilized to screen of potential ACP inhibitors, indicating that this probe has great potential in clinical practice.

A novel and sensitive ratiometric fluorescent quantum dot-based biosensor for alkaline phosphatase detection in biological samples via the inner-filter effect.

Alkaline phosphatase (ALP) is an important biomarker whose abnormal level in activity is associated with hepatobiliary, skeletal, and renal diseases as well as cancer. Herein, we synthesized ZnSe@ZnS quantum dots (ZnSe@ZnS QDs) and Mn-doped ZnS quantum dots (Mn:ZnS QDs) as fluorophores to establish the ratiometric fluorescent assay for ALP activity detection in biological samples. p-Nitrophenyl phosphate (PNPP) was used as a substrate for ALP, and the overlaps between absorption spectra of PNPP and excitation spectra of QDs resulted in sharp fluorescence quenching. Under the catalysis of ALP, PNPP was hydrolyzed into p-nitrophenol (PNP), which caused a red shift of absorption band of PNPP and fluorescence recovery of Mn:ZnS QDs (585 nm). However, the overlaps between absorption spectra of PNP and emission spectra of ZnSe@ZnS QDs led a further quenching of ZnSe@ZnS QDs (405 nm). Therefore, the ratiometric fluorescent signals (F 585/F 405) were associated with activity of ALP based on bidirectional responses of QDs to the concentration of PNPP. Under the optimum conditions, the method exhibited a good linear relationship from 4 to 96 U per L (R 2 = 0.9969) with the detection limit of 0.57 U per L. Moreover, the method was successfully applied for detecting the ALP activity in a complex biological matrix (human serum and HepG2 cells) with impressive specificity. In particular, the complicated chemical modifications of QDs and pretreatments of biological samples were not required in the whole detection procedures. Therefore, it not only provided a sensitive, specific and simple approach to clinical ALP activity detection, but it also provided support for early diagnosis of diseases.

A ratiometric fluorescent biosensor for rapid detection of Burkholderia pseudomallei by dual CRISPR/Cas12a trans-cleavage assisted signal enhancement

Melioidosis caused by Burkholderia pseudomallei (Bp) infection has high mortality in tropical and subtropical regions. A rapid and sensitive method for its detection is urgently needed. Herein, a ratiometric fluorescent sensor with dual recognition sites of target was designed for sensitive detection of Bp DNA. The sensor was constructed with a TAMRA-labeled DNA as probe, which could adsorb on polymer dots (Pdots) at acidic pH to produce TAMRA fluorescence at the excitation wavelength of Pdots via fluorescence resonance energy transfer (FRET). In the presence of target DNA, the trans-cleavage activity of CRISPR/Cas12a was activated with the dual recognition sites, which digested the probe DNA to weaken the FRET. With the fluorescence ratio of Pdots to TAMRA as the detection signal, a ratiometric fluorescent biosensing was thus achieved. This method with dual CRISPR/Cas12a trans-cleavage assisted signal enhancement showed a detectable range of target DNA from 1.0 pM to 10 nM, and could be successfully used to detect Bp DNA extract with a detection time of 40 min. The excellent performance such as high sensitivity, good specificity, acceptable accuracy and short analytical time along with convenient operation demonstrated its potential in clinic diagnosis of melioidosis.

Role of the Probe Sequence/Structure in Developing an Ultra-Efficient Label-Free COVID-19 Detection Method Based on Competitive Dual-Emission Ratiometric DNA-Templated Silver Nanoclusters as Single Fluorescent Probes.

We report the development of a label-, antibody-, enzyme-, and amplification-free ratiometric fluorescent biosensor for low-cost and rapid (less than 12 min) diagnosis of COVID-19 from isolated RNA samples. The biosensor is designed on the basis of cytosine-modified antisense oligonucleotides specific for either N gene or RdRP gene that can form silver nanoclusters (AgNCs) with both green and red emission on an oligonucleotide via a one-step synthesis process. The presence of the target RNA sequence of SARS-CoV-2 causes a dual-emission ratiometric signal transduction, resulting in a limit of detection of 0.30 to 10.0 nM and appropriate linear ranges with no need for any further amplification, fluorophore, or design with a special DNA fragment. With this strategy, five different ratiometric fluorescent probes are designed, and how the T/C ratio, the length of the stem region, and the number of cytosines in the loop structure and at the 3' end of the cluster-stabilizing template can affect the biosensor sensitivity is investigated. Furthermore, the effect of graphene oxide (GO) on the ratiometric behavior of nanoclusters is demonstrated and the concentration-/time-dependent new competitive mechanism between aggregation-caused quenching (ACQ) and aggregation-induced emission enhancement (AIE) for the developed ssDNA-AgNCs/GO nanohybrids is proposed. Finally, the performance of the designed ratiometric biosensor has been validated using the RNA extract obtained from more than 150 clinical samples, and the results have been confirmed by the FDA-approved reverse transcription-polymerase chain reaction (RT-PCR) diagnostic method. The diagnostic sensitivity and specificity of the best probe is more than >90%, with an area under the receiver operating characteristic (ROC) curve of 0.978.

A NIR ratiometric fluorescent biosensor for sensitive detection and imaging of α‐L‐fucosidase in living cells and HCC tumor‐bearing mice

AbstractDetection and imaging of α‐L‐fucosidase (AFU) is of great value to understand its roles in hepatocellular carcinoma (HCC) and tumor early diagnosis, but ideal assays are still lacking. Herein, a near‐infrared (NIR) fluorescent biosensor (α‐Fuc‐DCM) was elaborately designed and synthesized for rapid and ratiometric detection of AFU activity in cells and HCC tumor mouse models. In the presence of AFU, this biosensor shows an enhancement in NIR emission in a ratiometric manner, which significantly improves the detection accuracy with the limit of detection as low as 4.8 mU/mL. Taking advantage of these merits, the activity of AFU in lysosomes could be visualized using ratiometric and NIR dual modality in living cells. Furthermore, its remarkable application for monitoring of endogenous AFU activity in HCC tumor‐bearing mouse model is also demonstrated with bright fluorescence signal, which indicated that the biosensor could clearly monitor the liver tumor in the early stage. Importantly, the α‐Fuc‐DCM probe can be utilized to detect the AFU in serum from HCC patients. This strategy offers a promising biosensor system for early diagnosis of HCC and studying the roles of AFU in cancers.

Early calcium and cardiac contraction defects in a model of phospholamban R9C mutation in zebrafish

The phospholamban mutation Arg 9 to Cys (R9C) has been found to cause a dilated cardiomyopathy in humans and in transgenic mice, with ventricular dilation and premature death. Emerging evidence suggests that phospholamban R9C is a loss-of-function mutation with dominant negative effect on SERCA2a activity. We imaged calcium and cardiac contraction simultaneously in 3 and 9 days-post-fertilization (dpf) zebrafish larvae expressing plnbR9C in the heart to unveil the early pathological pathway that triggers the disease. We generated transgenic zebrafish lines expressing phospholamban wild-type (Tg(myl7:plnbwt)) and phospholamban R9C (Tg(myl7:plnbR9C)) in the heart of zebrafish. To measure calcium and cardiac contraction in 3 and 9 dpf larvae, Tg(myl7:plnbwt) and Tg(myl7:plnbR9C) fish were outcrossed with a transgenic line expressing the ratiometric fluorescent calcium biosensor mCyRFP1-GCaMP6f. We found that PlnbR9C raised calcium transient amplitude, induced positive inotropy and lusitropy, and blunted the β-adrenergic response to isoproterenol in 3 dpf larvae. These effects can be attributed to enhanced SERCA2a activity induced by the PlnbR9C mutation. In contrast, Tg(myl7:plnbR9C) larvae at 9 dpf exhibited ventricular dilation, systolic dysfunction and negative lusitropy, hallmarks of a dilated cardiomyopathy in humans. Importantly, N-acetyl-L-cysteine rescued this deleterious phenotype, suggesting that reactive oxygen species contribute to the pathological pathway. These results also imply that dysregulation of calcium homeostasis during embryo development contributes to the disease progression at later stages. Our in vivo model in zebrafish allows characterization of pathophysiological mechanisms leading to heart disease, and can be used for screening of potential therapeutical agents.

A ratiometric fluorescent aptasensor based on EXPAR to detect shellfish tropomyosin in food system

Shellfish allergy represents a crucial public health problem, resulting in an urgent requirement for an accurate, simple, and reliable method to quantify shellfish allergens in the complex food system. Tropomyosin (TM) is known as the major allergen in shellfish. Herein, a novel switch-conversional ratiometric fluorescent biosensor, using TM-specific aptamer as the recognition probe, was developed to detect TM in the complex food system. The proposed aptasensor exhibited good accuracy, stability, and selectivity for the quantification of TM in foodstuffs. Furthermore, the applicability of this proposed aptasensor in complex food samples was verified using seven different shellfish samples. Therefore, this ratiometric fluorescent aptasensor enables accurately detecting the presence of TM traces in foods, providing technical support for allergen label supervision.

A ratiometric fluorescent biosensor based on magnetic-assisted hybridization chain reaction and DNA-templated silver nanoclusters for sensitive microRNA detection

A biosensor was designed for sensitive detection of microRNA (miR)-144-3p, a potential miR biomarker for nasopharyngeal cancer, by integrating magnetic beads (MBs) into a hybridization chain reaction-silver nanoclusters (HCR-AgNCs) system. Through careful design, each component in the MBs-HCR-AgNCs biosensor was able to perform well without affecting subsequent steps. The target miR-144-3p was first magnetically separated with MBs coated with capture probes and short assistant probes. The collected miR was then amplified via HCR and detected using fluorogenic AgNCs that were prepared in situ. The DNA hairpins used in HCR were designed to host a AgNCs nucleation sequence that is capable of dual-emissive fluorescence response, thus enabling a ratiometric analysis. The novel MBs-HCR-AgNCs biosensor exhibits a good fluorescence response towards the target miR-144-3p with a wide linear detection range of 10 fM–1 μM and a detection limit of 4.88 fM. Moreover, it shows remarkable selectivity towards the target miR and good reproducibility even in raw human serum samples. Our findings suggest that the MBs-HCR-AgNCs can be a promising tool for sensitive miR detection in clinical diagnostics, without the need for complex sample preparation.

Ratiometric Fluorescent Biosensor Based on Self-Assembled Fluorescent Gold Nanoparticles and Duplex-Specific Nuclease-Assisted Signal Amplification for Sensitive Detection of Exosomal miRNA.

The sensitive detection of cancer-associated exosomal microRNAs shows enormous potential in cancer diagnosis. Herein, a ratiometric fluorescent biosensor based on self-assembled fluorescent gold nanoparticles (Au NPs) and duplex-specific nuclease (DSN)-assisted signal amplification was fabricated for sensitive detection of colorectal cancer (CRC)-associated exosomal miR-92a-3p. In this biosensing system, the hairpin DNA modified with sulfhydryl and fluorescent dye Atto-425 at both ends is conjugated to fluorescent Au NPs through Au-S bonds, resulting in the quenching of Atto-425. The miR-92a-3p can open the hairpin of DNA and forms an miR-92a-3p/DNA heteroduplex, triggering the specific cleavage of DSN for the DNA in the heteroduplex. As a result, Atto-425 leaves the fluorescent Au NPs and recovers the fluorescence emission. The released miR-92a-3p can hybridize with another hairpin DNA and lead to a stronger fluorescence recovery of Atto-425 to form a signal amplification cycle. The stable fluorescence of Au NPs and the changing fluorescence of Atto-425 constitute a ratiometric fluorescent system reflecting the concentration of miR-92a-3p. This biosensor exhibits excellent specificity and can distinguish CRC patients from healthy individuals by detecting miR-92a-3p extracted from clinical exosome samples, showing the potential in CRC diagnosis.

Ratiometric Fluorescent Metal-Organic Framework Biosensor for Ultrasensitive Detection of Acrylamide.

Acrylamide is a neurotoxin and carcinogen that forms during the thermal processing of food, inflicting irreversible harm to human health. Herein, a ratiometric fluorescence biosensor based on a 6-carboxyfluorescein-labeled aptamer (FAM-ssDNA) and porphyrin metal-organic framework (PCN-224) was developed. PCN-224 exhibits strong adsorption capacity for FAM-ssDNA and also quenches the fluorescence of FAM-ssDNA via fluorescence resonance energy transfer and photoinduced electron transfer. FAM-ssDNA hybridizes with complementary DNA to form double-stranded DNA (FAM-dsDNA), which is liberated from the PCN-224 surface, resulting in fluorescence recovery. However, the intrinsic fluorescence of the ligand remains unchanged. Acrylamide can create an adduct with FAM-ssDNA and inhibit the hybridization of FAM-dsDNA, thus realizing ratiometric sensing of acrylamide. The proposed biosensor displays excellent detection performance from 10 nM∼0.5 mM with a limit of detection of 1.9 nM. In conclusion, a fabricated biosensor was successfully applied to detect acrylamide in thermally processed food, and the results were consistent with those of high-performance liquid chromatography.

Ratiometric Fluorescence Detection of Colorectal Cancer-Associated Exosomal miR-92a-3p with DSN-Assisted Signal Amplification by a MWCNTs@Au NCs Nanoplatform.

The detection of miRNA shows great promise in disease diagnosis. In this work, a ratiometric fluorescent biosensor based on multi-walled carbon nanotubes@gold nanoclusters (MWCNTs@Au NCs) and duplex-specific nuclease (DSN)-assisted signal amplification was fabricated for miRNA detection. Colorectal cancer (CRC)-associated miR-92a-3p extracted from exosomes was selected as the target. MWCNTs@Au NCs performs the dual functions of fluorescence quencher and internal fluorescence reference. In the absence of miR-92a-3p, an Atto-425-modified single-stranded DNA probe is adsorbed on MWCNTs@Au NCs, resulting in the quenching of Atto-425. In the presence of miR-92a-3p, the duplex is formed by hybridization of the probe and miR-92a-3p and leaves the MWCNTs@Au NCs, resulting in the fluorescence recovery of Atto-425. DSN can cleave the probe and result in the release of miR-92a-3p. The released miR-92a-3p can hybridize with other probes to form a signal amplification cycle. The fluorescence of MWCNTs@Au NCs remains stable and constitutes a ratiometric fluorescence system with that of Atto-425. A detection concentration interval of 0.1–10 pM and a limit of detection of 31 fM was obtained under optimized measurement conditions. In addition, the accuracy of the biosensor was validated by detecting the concentration of miR-92a-3p extracted from clinical exosome samples.

A ratiometric fluorescent biosensor based on self-fluorescent MOF and target-triggered rolling circle amplification for sensitive detection of exosome-derived miRNA

MicroRNAs (miRNAs) are considered as biomarkers and display great potential in the diagnosis of diseases. As a colorectal cancer (CRC)-associated miRNA biomarker, miR-92a-3p possesses higher concentration in exosomes than in serum, causing it more feasible to diagnose CRC by measuring the concentration of miR-92a-3p inside exosomes. Herein, a rationally-engineered ratiometric fluorescent biosensor is proposed to detect the concentration of exosomal miR-92a-3p. The metal-organic framework (MOF-525) with self-fluorescence serves as both a fluorescent reference and a quencher of the fluorescence of single-stranded reporter by adsorption. The presence of miR-92a-3p triggers the looping of 5P-template strand and the further periodic rolling circle amplification. The periodic long strands and the reporters can form double strands to prevent the reporters from being adsorbed by MOF-525. The concentration of miR-92a-3p is positively correlated with the Δreporter/MOF-525 fluorescence intensity ratio. The biosensor exhibits a detection range of 0.1–10 pM and can differentiate miR-92a-3p from mismatched RNA sequences. The accuracy and practicality of the proposed biosensor for exosomal miRNA detection were evaluated by comparing with the conventional RT-qPCR. The as-obtained results are close to those of the RT-qPCR. This ratiometric fluorescent biosensor holds the potential for the sensitive detection of exosomal miRNA.

A ratiometric fluorescent aptamer homogeneous biosensor based on hairpin structure aptamer for AFB1 detection.

On the basis of aptamer (Apt) with hairpin structure and fluorescence resonance energy transfer (FRET), a ratio fluorescent aptamer homogeneous sensor was prepared for the determination of Aflatoxin B1 (AFB1). Initially, the Apt labeled simultaneously with Cy5, BHQ2, and cDNA labeled with Cy3 were formed a double-stranded DNA through complementary base pairing. The fluorescence signal of Cy3 and Cy5 were restored and quenched respectively. Thus, the ratio change of FCy3 to FCy5 was used to realized the detection of AFB1 with wider detection range and lower limit of detection (LOD). The response of the optimized protocol for AFB1 detection was wider linear range from 0.05 ng/mL to 100 ng/mL and the LOD was 12.6 pg/mL. The sensor designed in this strategy has the advantages of simple preparation and fast signal response. It has been used for the detection of AFB1 in labeled corn and wine, and has good potential for application in real samples.

Lanthanide doped metal-organic frameworks as a ratiometric fluorescence biosensor for visual and ultrasensitive detection of serotonin

It is of considerable importance to develop a fluorescence sensor with high efficiency and good sensitivity in biosensing. In this work, a ratiometric fluorescence sensor was constructed by incorporating Tb3+in situ into a bismuth-based metal-organic framework (Tb3+-NOTT-220) and subsequently utilized for rapid and sensitive detection of serotonin (5-hydroxytryptamine) in serum media. When serotonin was exposed to the sensor, the energy transfer from the ligand to Tb3+ was inhibited upon addition of serotonin, which is accompanied by the increase in the fluorescence intensity of the ligand and reduce the emission of Tb3+. Thus, a ratiometric fluorescence assay can be established by virtue of Tb3+ and ligand as the recognition site for serotonin detection. The developed sensor exhibited good selectivity and sensitivity to serotonin with the limit of detection of 0.57 ​μM and 1.21 ​μM in water and serum, respectively. Furthermore, the application of fluorescent hydrogel provides an effective scheme for rapid and portable determination of serotonin. The ratiometric fluorescence sensing system presents a new strategy for simple, reliable and on-site determination of serotonin.