Fluorescence Polarization Signal Research Articles

Click chemistry-based fluorescence polarization sensor for sensitive detection of ampicillin

Ampicillin (AMP) is a β-lactam antibiotic that can inhibit bacterial wall synthesis. The overuse and misuse of AMP makes it micropollutant that commonly found in food and various environmental media. In this work, a fluorescence polarization sensor was designed to sensitive detection of trace ampicillin based on click chemistry, using graphene oxide (GO) as a fluorescence polarization (FP) signal enhancement element. First, when ampicillin binds to its aptamer (apt), the adjacent alkyne and azide groups are separated, hindering the click-linking reaction. When Carboxyfluorescein (FAM) fluorophore-labeled probe (C-FAM) is added, its protruding 3-terminal FAM is recognized and cleaved by exonuclease I (EXO I), releasing fluorophores free that could not be adsorbed on GO, resulting in a lo0wer polarization signal. If there is no AMP in the system, aptamer probe is connected to its complementary chain ends by a click reaction. After C-FAM hybridizes with apt, the apt/P duplex is opened and the prominent single-stranded ends adsorb on the GO, leading a significantly enhanced FP signal. According to the relationship between the difference in FP values and the concentrations of AMP, the limit of detection of proposed method is as low as 80 pg/mL. This assay has a wide linear range plus excellent selectivity, and has been applied to detect AMP in milk and river water samples with satisfactory results, which demonstrates that the FP sensor has great potential for practical applications in food safety and environmental protection fields.

Recognition mechanism of split T-2 toxin aptamer coupled with reliable dual-mode detection in peanut and beer

T-2 toxin is a highly poisonous trichothecene and a well-known contaminant in cereal grains and cereal-based products, hence it is essential to design a simple, reliable, and precise detection mechanism for ensuring food safety and human health. In response to this imperative, we innovatively designed an unreported split T-2 toxin aptamer probe, and the cooperative binding mechanism of the split aptamer to T-2 was first investigated. Based on these, a dual-mode fluorescence (FL) and fluorescence polarization (FP) aptasensor was developed for T-2 toxin detection in peanut and beer samples. The results showed an excellent detection property of the aptasensor, indicating a linear range of 0.1–20 nM and 0.2–50 nM at a detection limit of 0.1 nM, 0.12 nM for FL and FP signals, respectively. In actual sample assays, recoveries of 101.7%–115.2%, 109.8%–121.6% for FL and FP mode, respectively, were reached in peanuts, whereas recoveries ranged from 81.0% -124.0%, 94.1%–107.8% for FL and FP mode in beer. This designed dual-mode biosensor not only showed excellent performances but also verified each other and proved the reliability of binary split aptamer molecular probes.

Fluorescence Polarization Assays for Organic Compounds in Food Safety.

Elevated concentrations of toxic organic compounds observed in food products pose serious dangers to human health. Both natural and artificial pollutants can cause food contamination. The stages of food production, packaging, transportation, and storage can also largely cause the appearance of undesirable substances in food products. The health consequences of ingesting food containing toxic contaminants range from mild gastroenteritis to deaths resulting from dysfunctional internal organs and neurological syndromes. The World Health Organization (WHO) sets recommendations for the content of such chemicals in food, including a minimum allowable concentration considered safe for human consumption. However, the control of food products from chemical pollutants is necessary. Moreover, fast, sensitive, and inexpensive methods are needed to detect them at the point of need. Currently, immune analysis methods are most widely used to determine pollutants in food. The development of fluorescence polarization immunoassay (FPIA) methods in a competitive format is a powerful and modern tool for detecting organic molecules in various matrices, thereby making FPIA methods useful for food safety applications. Due to the availability of portable devices for measuring the fluorescence polarization signal, FPIA methods can be used at the point of need. The variety of fluorescent labels and recognizing elements (receptors, monoclonal and polyclonal antibodies, and nanobodies) permits fluorescence polarization (FP) assays to detect significantly lower limits of organic substances. The FP assay is a homogeneous, fast, and quantitative method. The development of various formats of FP assays makes them promising in determining food pollutants. This review summarizes publications on FP analyses for detecting organic contaminants (pesticides, hormones, toxins, antibiotics, and other pharmaceuticals) in food products during 2018-2023. Further, it demonstrates the prospects for using this method to determine pollutants at the point of need and for detecting high molecular weight substances, fungi, and bacterial infections during food safety inspections.

Detection of Dibutyl Phthalate in Surface Water by Fluorescence Polarization Immunoassay.

Dibutyl phthalate (DBP) is widely used as a plasticizer in the production of polymeric materials to give them flexibility, strength and extensibility. However, due to its negative impact on human health, in particular reproductive functions and fetal development, the content of DBP must be controlled in food and the environment. The present study aims to develop a sensitive, fast and simple fluorescence polarization immunoassay (FPIA) using monoclonal antibodies derived against DBP (MAb-DBP) for its detection in open waters. New conjugates of DBP with various fluorescein derivatives were obtained and characterized: 5-aminomethylfluorescein (AMF) and dichlorotriazinylaminofluorescein (DTAF). The advantages of using the DBP-AMF conjugate in the FPIA method are shown, the kinetics of binding of this chemical with antibodies are studied, the analysis is optimized, and the concentration of monoclonal antibodies is selected for sensitivity analysis-16 nM. The calibration dependence of the fluorescence polarization signal for the detection of DBP was obtained. The observed IC50 (DBP concentration at which a 50% decrease in the fluorescence polarization signal occurs, 40 ng/mL) and the limit of detection (LOD, 7.5 ng/mL) values were improved by a factor of 45 over the previously described FPIA using polyclonal antibodies. This technique was tested by the recovery method, and the high percentage of DBP discovery in water ranged from 85 to 110%. Using the developed method, real water samples from Lake Onega were tested, and a good correlation was shown between the results of the determination of DBP by the FPIA method and GC-MS. Thus, the FPIA method developed in this work can be used to determine DBP in open-water reservoirs.

Cascade-amplified fluorescence polarization assay for miRNA based on aggregation strategy of Y-shaped DNA

MicroRNAs (miRNAs) have emerged as one of the promising biomarkers. Their aberrant levels are intimately connected to various diseases. However, Sensitive assay of low-abundance miRNAs remain a grand challenge. Herein, a novel sensitive cascade-amplified fluorescence polarization (FP) sensing platform for miRNA detection was developed based on aggregation strategy of Y-shaped DNA via streptavidin (SA). The miRNA-155 was employed as the model target analyte. In the presence of miRNA-155, the duplex-specific nuclease (DSN) cycling amplification and catalytic hairpin assembly (CHA) cycling amplification were triggered successively. Owing to the target-triggering dual cyclic amplification, numerous Y-shaped DNA nanostructure could be easily generated according to the assembly order of hairpin probes. Then, SA was added to the system, inducing the aggregation of Y-shaped DNA nanostructure to form a large-size of DNA/SA complexes. Taking advantages of cascade-amplified strategy, a significant enhancement of the FP signal was achieved and a sensitive assay for miRNA detection was realized with the detection limit of 1 × 10-13 M. Beyond that, the proposed method could distinguish single-base mismatch and achieved satisfactory result for the expression level assay of miRNA-155 in different cell lines, holding great potential in the applications in miRNA-related clinical diagnosis and biochemical researches.

Amplification of fluorescence polarization signal based on specific recognition of aptamers combined with quantum quenching effect for ultrasensitive and simple detection of PCB-77

Here, we report a novel aptasensor based on decahedral silver nanoparticles (Ag10NPs) enhanced fluorescence polarization (FP) for detecting PCB-77. Using aptamer modified Ag10NPs hybridized with DNA sequence labeled fluorescent group as an analytical probe, polychlorinated biphenyls (PCB-77) could be detected with high sensitivity and selectivity. The linear range of determination was 0.02 ng/L to 390 ng/L and the limit of detection was 5 pg/L. In addition, through the optimization of the experiment condition and signal probe DNA (pDNA), we found that the maximum FP signal could be generated when the distance between fluorescein and the surface of Ag10NPs was 3 nm. When the aptamer was immobilized on the surface of Ag10NPs could be strengthened the anti-interference performance of aptamer nanoprobe and further improved the detection ability. At the same time, we also compared the detection performance of the traditional FP signal enhancer streptavidin (SA) analysis system. The fluorescence polarization aptasensor could detect PCB-77 samples efficiently in complex environmental water, which shows a good application prospect.

Selection, truncation and fluorescence polarization based aptasensor for Weissella viridescens detection

Weissella viridescens is a spoilage bacterium commonly found in low-temperature meat products. In this work, after fifteen rounds including three counter selection rounds of whole-cell systemic evolution of ligands by exponential enrichment (SELEX) in vitro, a novel aptamer L3 that can specifically recognize W. viridescens was obtained with a dissociation constant (Kd) value of 68.25 ± 5.32 nM. The sequence of aptamer L3 was optimized by truncation and a new aptamer sequence TL43 was obtained with a lower Kd value of 32.11 ± 3.01 nM. Finally, a simple and rapid fluorescence polarization (FP) platform was constructed to detect W. viridescens, in which FAM-labeled complementary sequence (FAM-cDNA) was employed to generate FP signal and streptavidin was used to amplify FP signal. In the presence of target bacteria, FP value decreased owning to the dissociation of FAM-cDNA from streptavidin/biotin-TL43/FAM-cDNA complex. Under optimal conditions, the concentration of W. viridescens and FP value displayed a good linear relationship with the detection range from 102 to 106 cfu/mL. Moreover, the designed detection system had a good recovery rate of 90.6%–107.7% in smoked ham samples compared with classical plate counting method, indicating the great potential of the selected and truncated aptamer in practical biosensing applications.

Performance improved fluorescence polarization for easy and accurate authentication of chicken adulteration

Rapid, accurate, and effective authentication of unlabeled meat components in commercial meat products is of great significance for guaranteeing food safety and human health. In this research, we have constructed a single-strand binding protein (SSB)-assisted performance improved fluorescence polarization (FP) protocol for authentication of chicken component in meat products. The SSB-assisted FP strategy involves the use of fluorescein isothiocyanate (FITC)-labeled primers to perform polymerase chain reaction (PCR). In addition, FP enhancement was accomplished by mixing the amplified product with SSB. Based on the designed mechanism, the free primers in the final amplified product can be tightly bound by SSB, which greatly restricts the rotation of FITC, thereby resulting a high FP signal. In the presence of chicken DNA, the transformation of single-stranded primers to double-stranded amplicons greatly hinders the combination of SSB and primers, leading to the decrease of FP signal. This method well couples the advantages of both PCR and FP technology. Through this new strategy, as low as 0.035% (wt. %) of chicken adulteration in meat samples can be well authenticated. Moreover, this method is also available for commercial meat products. We expect this SSB-assisted FP strategy will provide new insights into the construction of versatile analysis tools in the field of food safety detection. • Fluorescence polarization protocol for rapid sensing of chicken adulteration. • Fluorescence polarization adopted for analysis of PCR products. • Single strand binding protein adopted to improve detection limit of adulteration. • Successful authentication of commercial meat products with improved FP strategy.

An amplified fluorescence polarization assay for sensitive sensing of organophosphorus pesticides via MnO2 nanosheets

It is highly desirable to develop a simple, efficient and sensitive strategy for organophosphorus pesticides (OPs) in both environment pollution and human health. Herein, a novel amplified fluorescence polarization (FP) biosensor was established for highly sensitive detection of OPs using MnO2 nanosheets as the signal enhancer. In this system, OPs can suppress the activity of acetylcholinesterase (AChE) efficiently, blocking the hydrolysis reaction of acetylthiocholine (ATCh) to generate thiocholine (TCh) by AChE. TCh can lead the decomposition of MnO2 nanosheets to manganese ions. So, without the influence of TCh, MnO2 nanosheets can maintain its original shape and form a stable complex with FAM-DNA, which greatly enhanced the FP signal. This method can tremendously improve the sensitivity of FP with a detection limit of 0.01 ng/mL for diazinon. In addition, it was also applicable to determine other four OPs and investigate the level of diazinon in real water samples. Consequently, the proposed approach provides a new promising platform for detection of OPs and is expected to be used in application of environmental monitoring.

Framework nucleic acid-wrapped protein-inorganic hybrid nanoflowers with three-stage amplified fluorescence polarization for terminal deoxynucleotidyl transferase activity biosensing

Herein, we proposed a terminal deoxynucleotidyl transferase (TdT), a potential biomarker of lymphoid tumors, responsive fluorescence polarization (FP)- sensing protocol based on framework nucleic acid (FNA)-wrapped protein-inorganic hybrid nanoflowers. To achieve this goal, a pair of poly-A-composed extension primers (EPa and EPb) was designed, and protein-inorganic hybrid nanoflowers were synthesized by a biomineralization reaction. EPa was labeled with carboxyfluorescein (FAM) fluorophore to create the preliminary FP signal. EPb was labeled with biotin to conjugate with hybrid nanoflowers. Upon introduction of TdT into the dTTP pool, both EPa and EPb can be catalyzed by TdT to incorporate numerous T bases, thereby facilitating intermolecular hybridization between ‘A’ and ‘T’ bases. The final assembled FNA-wrapped hybrid nanoflowers with greatly enhanced molecular volume and weight restrict the free rotation of attached FAMs, causing a great FP enhancement from a designated three-stage FP amplification. Under optimized conditions, the TdT can be detected with a detection limit of 0.023 U/mL and a linear detection from 0.1 U/mL to 100 U/mL within 20 min. As a proof-of-concept study, the first exploitation of FNA and protein-inorganic nanoflowers to improve the FP signal and the merit of FP without sample separation and washing opens a new avenue for biochemical study and disease diagnosis.

Combining single-molecule super-resolved localization microscopy with fluorescence polarization imaging to study cellular processes

Super-resolution microscopy has catalyzed valuable insights into the sub-cellular, mechanistic details of many different biological processes across a wide range of cell types. Fluorescence polarization spectroscopy tools have also enabled important insights into cellular processes through identifying orientational changes of biological molecules typically at an ensemble level. Here, we combine these two biophysical methodologies in a single home-made instrument to enable the simultaneous detection of orthogonal fluorescence polarization signals from single fluorescent protein molecules used as common reporters on the localization of proteins in cellular processes. These enable measurement of spatial location to a super-resolved precision better than the diffraction-limited optical resolution, as well as estimation of molecular stoichiometry based on the brightness of individual fluorophores. In this innovation we have adapted a millisecond timescale microscope used for single-molecule detection to enable splitting of fluorescence polarization emissions into two separate imaging channels for s- and p-polarization signals, which are imaged onto separate halves of the same high sensitivity back-illuminated CMOS camera detector. We applied this fluorescence polarization super-resolved imaging modality to a range of test fluorescent samples relevant to the study of biological processes, including purified monomeric green fluorescent protein, single combed DNA molecules, and protein assemblies and complexes from live Escherichia coli and Saccharomyces cerevisiae cells. Our findings are qualitative but demonstrate promise in showing how fluorescence polarization and super-resolved localization microscopy can be combined on the same sample to enable simultaneous measurements of polarization and stoichiometry of tracked molecular complexes, as well as the translational diffusion coefficient.

Label free structure-switching fluorescence polarization detection of chloramphenicol with truncated aptamer

In this study, the original chloramphenicol aptamer containing 80 bases was truncated to 30 bases with high affinity by the SYBR Green I assay. It was found that the ionic strength and type affect the recognition of aptamers, especially magnesium ion played a vital role in the binding process. Furthermore, the binding performance of aptamer, including binding mode, key binding sites and conformational changes were further investigated by circular dichroism spectroscopy, UV–vis absorption spectrum and molecular docking. Based on these research data, we inferred that chloramphenicol bound to the minor groove region in the aptamer double helix. Finally, the optimized aptamer LLR10 was used to develop a novel label free fluorescence polarization assay to detect chloramphenicol within SYBR Green I as the source of fluorescence polarization signal. Under optimal conditions, the designed method showed a linear detection range of 0.1–10 nM with a detection limit of 0.06 nM. Additionally, the aptasensor exhibited a high accuracy to the detection of chloramphenicol in milk samples with a recovery rate from 93.7% to 98.4%. Therefore, the developed label free fluorescence polarization aptasensor provides a new idea for the rapid, reliable and sensitive detection of chloramphenicol, which can be applied to food safety control.

A fluorescence polarization aptasensor coupled with polymerase chain reaction and streptavidin for chloramphenicol detection

The authors describe a fluorescence polarization (FP) aptasensor based on the polymerase chain reaction (PCR) and streptavidin as dual FP amplifiers to detect chloramphenicol residues in food. Briefly, label-free aptamer was incubated with chloramphenicol and the aptamer-chloramphenicol conjugate was used as a template. Subsequently, the FAM-labeled forward primer and biotin-labeled reverse primer were added for PCR to amplify the template and the FAM-labeled primer. The molecular weight of FAM-labeled primer increased rapidly and the corresponding FP also enhanced. Finally, with the introduction of streptavidin, the PCR products and streptavidin were combined with the biotin-streptavidin interactions, resulting in much larger molecular weight. Thus, a dual amplified FP signal was obtained. Under optimal conditions, we were able to achieve a wide linear detection range of 0.001–200 nM. In addition, the designed strategy was applied to detect chloramphenicol in honey samples with high accuracy. Moreover, the strategy can be easily extended to detect other small molecules by changing the corresponding aptamers, which provide a promising avenue for the detection of small molecules by FP.

A T7 exonuclease assisted dual-cycle signal amplification assay of miRNA using nanospheres-enhanced fluorescence polarization

Based on streptavidin coated nanospheres and T7 exonuclease assisted dual-cycle signal amplification, we developed a novel sensitive fluorescence polarization detection method for miRNA. When target miRNA was present in the system, hairpin probe hybridized with miRNA, forming a double-stranded structure. The 5′ end of hairpin probe was then recognized and digested by T7 exonuclease, releasing the non-degraded single strand DNA fragments and miRNA. The released target miRNA could trigger the next cycle of hybridization and digestion, releasing more non-degraded fragments from hairpin probe. The fragments could hybridize with a signal probe (with carboxyfluorescein modification at 5′-end and biotin modification at 3′-end). The formed blunt 5′-end of signal probe was then recognized and degraded by T7 exonuclease, releasing the fragments and the fluorophore carboxyfluorescein. The next cycle of hybridization and digestion of signal probe was triggered by the released fragment at the same time. The free carboxyfluorescein cannot connect with streptavidin coated nanospheres which were used as the fluorescence polarization signal amplifier. So, there was a big change of fluorescence polarization signal after adding miRNA into the detection system, due to the different fluorescence polarization signal between nanospheres-captured intact signal probe and free carboxyfluorescein. The detection limit of this method is about 0.001 nM, and it has a good selectivity. In addition, it was also applicable to determine target miRNA in total miRNA extracts and compare the expression level of target miRNA in different cells. Consequently, the proposed method is expected to be used for the potential cancer diagnosis and the related biomedical research.

Fluorescence polarization sensor platform based on gold nanoparticles for the efficient detection of Ag (І)

Abstract Silver is one of the most important heavy metals with a broad range of applications; however, it can have significant negative impacts on both the ambient environment and organisms. For this work, an interesting fluorescence polarization sensor based on SH-DNA functionalized gold nanoparticles (AuNPs) was proposed for the detection of Ag+ in aqueous solutions. Moreover, this sensing strategy is quite simple, sensitive, selective, and cost-effective. Ag+ ions can interact specifically with a cytosine–cytosine (C–C) mismatch in DNA duplexes to form stable metal-mediated cytosine–Ag+–cytosine (C–Ag+–C) base pairs. The creation of these C–Ag+–C complexes results in evident changes in the molecular volume and fluorescence polarization signal. The stable hybridization between the two probes occurs with the formation of the C–Ag+–C complex in the presence of Ag+ ions, which leads to obvious fluorescence quenching in contrast to the system without AuNP. This assay may be employed to identify nanomolar levels of Ag+ within 6 min at room temperature. Furthermore, the sensor was successfully adopted to detect Ag+ ions in environmental water samples, which may facilitate the potential development of a range of environmental monitoring applications.

Chimeric Aptamers-Based and MoS2 Nanosheet-Enhanced Label-Free Fluorescence Polarization Strategy for Adenosine Triphosphate Detection.

Adenosine triphosphate (ATP) as a primary energy source plays a unique role in the regulation of all cellular events. The necessity to detect ATP requires sensitive and accurate quantitative analytical strategies. Herein, we present our study of developing a MoS2 nanosheet-enhanced aptasensor for fluorescence polarization-based ATP detection. A bifunctional DNA strand was designed to consist of chimeric aptamers that recognize and capture ATP and berberine, a fluorescence enhancer. In the absence of ATP, the DNA strand bound to berberine will be hydrolyzed when Exonuclease I (Exo I) is introduced, releasing berberine as a result. In contrast, when ATP is present, ATP aptamer folds into a G-quadruplex structure; thus, the complex can resist degradation by Exo I to maintain berberine for fluorescent detection purpose. In addition, to magnify the fluorescence polarization (FP) signal, MoS2 nanosheets were also adopted in the system. This nanosheets-enhanced FP strategy is simple and facile which does not require traditional dye-labeled DNA strands and complex operation steps. The developed fluorescence polarization aptasensor showed high sensitivity for the quantification of ATP with a detection limit of 34.4 nM, performing well both in buffer solution and in biological samples.

Magnetic nanoparticles and polydopamine amplified FP aptasensor for the highly sensitive detection of rHuEPO-α

In this paper, an amplified fluorescence polarization (FP) aptasensor based on magnetic nanoparticles @polydopamine (MNP@PDA) was innovatively developed for sensitive detection of recombinant human erythropoietin-alpha(rHuEPO-α). The amplified FP signal was due to the large mass of protein and MNP@PDA. And this assay can be utilized for target separation or recycling based on the magnetic property of MNP@PDA through magnetic separation. Briefly, rHuEPO-α and MNP@PDA were added successively to react with the labeled aptamer (FAM-P1), which both contributed to the increase of FP signal via the formation of FAM-P1-rHuEPO-α and particularly FAM-P1-MNP@PDA complex. The strong interaction between MNP@PDA and FAM-P1 ensured the high efficiency of mass amplification and magnetic separation. As a result, the detection limit for rHuEPO-α was 0.12 pM, 4 orders of magnitude lower than original assay. Besides, three kinds of rHuEPO-α injections, NuPIAO, Epogen and ESPO were used to evaluate the selectivity of this assay in complex matrix with reasonable standard deviation. In a word, this work provides a simple, rapid, non-modified, highly sensitive and selective sensing platform for the detection of rHuEPO-α.

Highly Simple and Sensitive Molecular Amplification-Integrated Fluorescence Anisotropy for Rapid and On-Site Identification of Adulterated Beef.

Fluorescence polarization (FP) signal is a self-referencing fluorescence signal, and it is less dependent on dye concentration and environmental interferences, which makes FP measurement a highly attractive alternative sensing technology to conventional fluorescent detection methods. Here we adopted a strategy for rapid increase of molecular weight to increase the FP signal for the detection of meat adulteration. The molecular weight of fluorescent labeled primers increased rapidly by slight preamplification, and the FP values were varied accordingly. We found a positive correlation between adulteration ratio and the FP signals. Detection limit for adulterated beef can be reached as low as 0.1% (wt %), meeting or better than the most detection requirements. On the basis of this proposed amplification-integrated FP method, both the standard samples and the commercial processed beef samples were successfully authenticated with satisfied results.

DNA-functionalized gold nanoparticle-based fluorescence polarization for the sensitive detection of silver ions.

Despite their practical applications, Ag+ ions are environmental pollutants and affect human health. So the effective detection methods of Ag+ ions are imperative. Herein, we developed a simple, sensitive, selective, and cost-effective fluorescence polarization sensor for Ag+ detection in aqueous solution using thiol-DNA-functionalized gold nanoparticles (AuNPs). In this sensing strategy, Ag+ ions can specifically interact with a cytosine-cytosine (CC) mismatch in DNA duplexes and form stable metal-mediated cytosine-Ag+-cytosine (C-Ag+-C) base pairs. The formation of the C-Ag+-C complex results in evident changes in the molecular volume and fluorescence polarization signal. To achieve our aims, we prepared two complementary DNA strands containing C-base mismatches (probe A: 5'-SH-A10-TACCACTCCTCAC-3' and probe B: 5'-TCCTCACCAGTCCTA-FAM-3'). The stable hybridization between probe A and probe B occurs with the formation of the C-Ag+-C complex in the presence of Ag+ ions, leading to obvious fluorescence quenching in comparison to the system without AuNP enhancement. The assay can be used to identify nanomolar levels of Ag+ within 6 min at room temperature, and has extremely high specificity for Ag+, even in the presence of higher concentrations of interfering metal ions. Furthermore, the sensor was successfully applied to the detection of Ag+ ions in environmental water samples and showed excellent selectivity and high sensitivity, implying its promising application in the future.

Graphene oxide-enhanced and proflavine-probed fluorescence polarization biosensor for ligand-RNA interaction assay

Proflavine could recognise HIV RRE (Rev response element) RNA, thus inhibiting its interaction with Rev peptide. Owing to the non-significant difference in fluorescence polarization (FP) signal between the proflavine and proflavine-RRE complex, it is not practicable for the identification of antagonists of HIV Rev peptide using proflavine as a FP probe. In the present study, graphene oxide (GO) was introduced to amplify the FP signal to enhance the sensitivity for the identification of Rev antagonists, thereby enabling the development of a primary GO-enhanced FP biosensor for the discovery of Rev antagonists. The effect of the amount of GO on the FP changes was investigated via displacement assay using a known Rev antagonist, neomycin B. The mechanism underlying the changes in FP caused by the addition of neomycin B was explored in the presence of GO. Finally, the performance of this biosensor was further evaluated using other 4 test drugs. The introduction of GO to this interaction system increased the practicability for the discovery of Rev antagonists using proflavine as a probe, and this label-free method does not suffer from limitations caused by fluorescent covalent labeling, thus providing a novel strategy for the characterization of ligand-RNA binding interactions.