Ratiometric Strategy Research Articles

Specifically triggered dissociation based ratiometric electrochemical sensor for H2O2 measurement in food samples

A novel ratiometric strategy based electrochemical sensor was developed to quantitative assay of H2O2 in different food samples. 4-aminophenylboronic acid pinacol ester (ABAPE) dissociation was specifically triggered by H2O2 to generate electro-active 4-aminophenol (4-AP), which not only can be oxidized to indirectly indicate the concentration of H2O2, but also endowed the sensor with high selectivity. Meanwhile, a reference probe of poly(thionine) (TH) was modified with ketjen black (KB) and gold nanoparticles (AuNPs) on electrode surface. KB and AuNPs displayed high electrocatalytic activity to 4-AP. A current ratio between 4-AP and TH (i/iTH) showed a good linear relationship with the concentration of H2O2 in a range of 3.0 × 10−7 – 1.0 × 10−4 mol/L (0.010 ppm − 3.40 ppm) with a limit of detection of 2.6 × 10−7 mol/L (0.009 ppm) (S/N = 3). Moreover, the ratiometric strategy based sensor possessed good accuracy, reliability, and stability, and successfully determined H2O2 in food samples with satisfactory results.

A smartphone integrated ratiometric fluorescent sensor for point-of-care testing of fluoride ions.

In view of the high toxicity and widespread availability, it is of great importance to develop accurate, sensitive, and convenient assays for fluoride ion (F-) detection. Herein, a ratiometric fluorescent system is established for point-of-care testing (POCT) of F- with a smartphone as analyzer. The sensing system of calcein-QDs-Eu3+ contains two fluorescent probes of calcein (green emission) and ZnCdSe/ZnS QDs (red emission). The sensing system only presents red emission in that the calcein emission is quenched due to the combination between calcein and Eu3+. When F- is introduced, the fluorescence of calcein is recovered due to the stronger interaction between F- and Eu3+, which changes the emission from red to green. The ratiometric strategy offers an obvious fluorescence color change of the system, which eliminates interference and improves the detection accuracy. Specifically, the sensing system has excellent selectivity in that Eu3+ is more inclined to bind with F- rather than other anions. The developed assay was further used to prepare a test paper and hydrogel for POCT. To further improve the detection sensitivity and realize quantitative analysis, a smartphone installed with a color scan app is integrated as signal reader and analyzer, which is used for POCT of F- in real samples, showing great application potential in environmental protection and food safety evaluation.

Magnetic antifouling material based ratiometric electrochemical biosensor for the accurate detection of CEA in clinical serum

A novel ratio electrochemical biosensor based on multi-functional nanocomposite was developed. Fe3O4 was synthesized in situ on carboxyl functionalized 2D nanomaterial MXene, and then covalently bonded with [Ru(NH3)6]3+ to obtain nanocomposites MXC-Fe3O4-Ru. Fe3O4 and [Ru(NH3)6]3+ can neutralize the electronegativity of the MXene to make the nanocomposites electrically neutral. Combine with the good hydrophilicity and conductivity of MXene, the nanocomposites can be utilized to construct antifouling electrochemical biosensors without modifying with specific antifouling materials. Moreover, Fe3O4 can endow the nanocomposites with magnetism, and [Ru(NH3)6]3+ is used as an internal standard molecule. The strong magnetic MXC-Fe3O4-Ru can be easily separated and firmly modified on the magnetic gold electrode (MGE). DNA double-stranded (dsDNA) containing an ferrocene (Fc)-modified carcinoembryonic antigen (CEA) aptamer can be specifically captured to the surface of the electrode by amido bond. In the presence of CEA, CEA binds to the aptamer and leaves the electrode surface, the electrochemical signal of Fc decreases, while the electrochemical signal of [Ru(NH3)6]3+ is fixed on the electrode surface remains basically unchanged. The ratio of the electrochemical signals of Fc and [Ru(NH3)6]3+ is proportional to the CEA concentration. The linear range of the sensor is 1 pg/mL to 1 μg/mL with a detection limit of 0.62 pg/mL. With the excellent antifouling performance, good conductivity of the nanocomposite, and the application of the ratiometric strategy, the biosensor can achieve high selectivity, accuracy, and sensitivity for the detection of targets even in complex samples, such as FBS and clinical serum.

A Novel Ratiometric Electrochemical Biosensor Using Only One Signal Tag for Highly Reliable and Ultrasensitive Detection of miRNA-21.

Herein, a novel ratiometric electrochemical biosensor with methylene blue (MB) as the only one signal tag was proposed for highly reliable and ultrasensitive detection of microRNA-21 (miRNA-21) under the assistance of an intelligent target-induced dual signal amplification (T-DSA). First, a small amount of target miRNA-21 could produce abundant mimic targets DNA S1 and Zn2+ through target-induced recycle and acid dissolution, respectively. Then, S1 triggered rolling circle amplification (RCA) to generate functional DNA nanospheres (DSP) encoded by DNAzyme and substrate sequence for loading numerous signal tag MB with a remarkable electrochemical signal (signal on), and the Zn2+ cofactor mediated the nonviolent DNAzyme-catalyzed cleavage of DSP to sharply release MB with obviously reduced electrochemical responses (signal off). Impressively, our strategy could controllably load and release the only signal tag MB through the well-designed DSP to effectively avoid the false positive responses caused by the non-ideal upright state of DNA probes connected to electrodes in traditional distance-dependent signal adjustment ratiometric strategies with two different signal tags. Meanwhile, with the aid of innovative T-DSA recycle and RCA-produced functional DSP, the detection sensitivity of this sensing platform was significantly improved. As a result, the proposed biosensor successfully realized highly reliable and ultrasensitive detection of miRNA-21 with a detection limit down to 26.7 aM, which shows exceptional promise in biological analysis and medical diagnosis.

A Cell-Anchored and Self-Calibrated DNA Nanoplatform for in Situ Imaging and Quantification of Endogenous MicroRNA in Live Cells: Introducing Two Controls to Normalize the Sensing Signals

A Cell-Anchored and Self-Calibrated DNA Nanoplatform for in Situ Imaging and Quantification of Endogenous MicroRNA in Live Cells: Introducing Two Controls to Normalize the Sensing Signals

An Iron(III)-Based Metal-Organic Gel-Catalyzed Dual Electrochemiluminescence System for Cytosensing and In Situ Evaluation of the VEGF165 Subtype.

The recent surge of interest in metal-organic gels (MOGs) has emerged for their soft porous structure, large surface area, and abundant active metal sites, making them a promising candidate for building catalyst matrices. In this work, facilely synthesized Fe(III)-organic gel was directly used as a robust electrode matrix. Detailed studies illustrated that their Fe(III) centers can speed up the electro-oxidation/reduction of the H2O2 coreactant to produce reactive oxygen species for enhancing a potential-resolved dual electrochemiluminescence (ECL) emission. Among them, the anodic signal of luminol varied with the cell concentration based on the impedance ECL mechanism, while the cathodic signal of CdS quantum dots traced the VEGF165 subtype at cell surface by specific aptamer recognition. Based on this, a ratiometric strategy was proposed for accurate cytosensing by eliminating environmental interference. Moreover, by cooperating these two signals, a novel strategy was developed for direct evaluation of the VEGF165 subtype, further realizing rapid drug screening and subtype assessment on different cell lines. This work not only opens up the promising application of MOGs as an effective catalyst matrix but also develops reliable cell assays and protein subtype identification for clinical diagnosis and research.

Aptamer-Target Recognition-Promoted Ratiometric Electrochemical Strategy for Evaluating the Microcystin-LR Residue in Fish without Interferences.

Given the significance of food safety, it is highly urgent to develop a sensitive yet reliable sensor for the practical analysis of algal toxins. As most of the developed sensors are disturbed by interfering substances and the target toxin is detected in a single-signal manner based on the immunoassay technology. Herein, we developed an aptamer-based dual-signal ratiometric electrochemical sensor for the sensitive and accurate analysis of microcystin-LR (MC-LR), using it as a proof-of-concept analyte. Methylene blue-tagged ssDNA (MB-ssDNA) was immobilized at the gold electrode surface accompanied with the absence of ferrocene-tagged ssDNA (Fc-ssDNA), resulting in a high differential pulse voltammetry (DPV) current of MB and a low DPV current of Fc. The recognition of MB-ssDNA by MC-LR stimulated the formation of MC-LR@MB-ssDNA, which induced the removal of MB-ssDNA from the electrode and the exposure of SH-ssDNA, enabling Fc-ssDNA to be captured at the electrode surface via nucleic acid hybridization. In comparison with MC-LR deficiency, the DPV signal of MB dropped along with an improved DPV signal of Fc, contributing to the ratiometric detection of MC-LR, with the limit of detection down to 0.0015 nM. Furthermore, this ratiometric electrochemical sensor was successfully explored to assess the bioaccumulated amount of MC-LR in the liver and meat of fish. The aptamer-based ratiometric strategy to develop an electrochemical MC-LR assay will offer a promising avenue to develop high-performance sensors, and the sensor will find more useful application in MC-LR-related aquatic product safety studies.

A nanozyme-catalysis-based ratiometric electrochemical sensor for general detection of Cd2.

A new kind of internal reference probe was developed to construct a ratiometric electrochemical sensor for accurate and sensitive Cd2+ detection. AuPt nanoclusters (AuPt NCs) were synthesized and then evenly distributed on reduced graphene oxide (rGO) to form a nanocomposite (AuPt-rGO), which showed peroxidase-like activity and catalyzed the oxidation of o-phenylenediamine (OPD) to 2,3-diaminophenazine (DAP) in the presence of H2O2. It was found that DAP was further reduced on the electrode with a constant current intensity whether Cd2+ was added or not, which enabled it to function as an excellent internal reference probe. Thus, the ratio between the reduction currents of Cd2+ and DAP (iCd2+/iDAP) was proposed to monitor Cd2+ based on the ratiometric strategy. Additionally, the high surface area and good conductivity of AuPt-rGO endowed the sensor with enhanced sensitivity. A wide linear range and a low limit of detection were measured at 5.0 × 10-8-1.0 × 10-4 mol L-1 and 3.1 × 10-8 mol L-1, respectively. Compared with the single signal sensors, this ratiometric sensor presents more accuracy and reliability in the detection of Cd2+, facilitating the quantification of Cd2+ concentration in different water samples with good recoveries. The novel internal reference probe proposed here shows great possibilities for accurate and sensitive Cd2+ detection based on the ratiometric strategy.

Polypeptide induced perylene probe excimer formation and its application in the noncovalent ratiometric detection of matrix metalloproteinase activity.

Matrix metalloproteinases (MMPs) are important biomarkers for a number of diseases. Thus, the precise determination of MMP activity is of crucial importance. Herein, we report a ratiometric fluorescence method for the sensitive and selective sensing of MMP activity. A number of positively charged MMP substrates (polypeptides) were designed and prepared. These polypeptides could induce aggregation of a negatively charged perylene diimide derivative (PC1). As a result, excimer fluorescence of PC1 was observed. Addition of the corresponding MMP resulted in cleavage of the polypeptide chain and dis-aggregation of PC1, which led to turning on of the PC1 monomer fluorescence. Based on the ratio of the monomer (545 nm, IM) and the excimer (680 nm, IM) fluorescence intensity changes, a ratiometric method I545/I680) was established to detect MMP activity. The enzymatic activity of a number of MMPs (MMP-1, 2, 3, 7, 9 and 13) could be determined with a limit of detection of 4.8, 2.2, 16, 6.0, 1.7 and 5.5 ng mL-1, respectively. Using MMP-2 and MMP-9 as examples, flavonoid herbal extracts as potential inhibitors were studied. It was observed that mangiferin, apigenin, quercetin and isoliquiritigenin had significant inhibiting effects on the enzyme activity. And these herbal extracts also inhibited tumor cell metastasis. Moreover, the developed strategy was also employed to determine the concentration of MMP-9 in human saliva samples. Since the method relies on only noncovalent interactions between the polypeptide and PC1, no covalent labeling of fluorescence dye on the polypeptide substrate is required, and the method is thus simple, broad-spectrum inexpensive and effective. It has the potential to be developed into a clinical test kit.

Highly Stable Electrochemical Probe with Bidentate Thiols for Ratiometric Monitoring of Endogenous Polysulfide in Living Mouse Brains.

The lack of reliable approaches for real-time measurement and quantification of polysulfides (H2Sn) in vivo greatly limits the exploration of their potential roles in brain functions. Herein, an electrochemical probe, 4-(5-(1,2-dithiolan-3-yl)pentanamido)-1,2-phenylene bis(2-fluoro-5-nitrobenzoate) (FP2), was rationally designed and created for determination of H2Sn. The bis-electrophilic groups of FP2 could specifically recognize two -SH groups in H2Sn and trigger the generation of an electroactive pyrocatechol moiety, resulting in a well-defined faradic current signal at ∼0.24 V (vs Ag/AgCl). Meanwhile, bidentate thiols were designed as anchoring sites to greatly improve the assembled stability of FP2 at the Au surface, which efficiently defended the interference of glutathione (GSH) with a current decrease of less than 5.2% even after long-term measurements in 5 mM GSH for 3 h. In addition, a stable inner reference molecule with dithiols, α-lipoic acid ferrocenylamide (FcBT), was synthesized to construct a ratiometric electrochemical strategy for in vivo determination of H2Sn through one-step coassembling with FP2 via double S-Au bonds. The present ratiometric strategy demonstrated high selectivity for real-time tracking of H2Sn in a linear range of 0.25-20 μM. Eventually, the developed microelectrode with high selectivity, accuracy, and stability was employed for in vivo assaying of H2Sn in mouse brains with ischemia.

Amplifiable ratiometric fluorescence biosensing of nanosilver multiclusters populated in three-way-junction DNA branches

To explore the fluorescence bio-responsiveness of emissive silver nanoclusters (AgNCs) populated in DNA-branched scaffolds is intriguing yet challenging. In response to a desired targeting model (T*) as a vehicle, herein a customized three-way-junction DNA construct (TWJDC) is assembled via competitive hybridizing cascade among three stem-loop hairpins with specific base sequences, where the repeated recycling of T* enables the exponentially amplifiable output of rigid TWJDC. As designed, these stable hybridization products are highly T*-stimulated responsive and constructing-directional. In the three branched-arms, the unpaired sticky ends provide isotropic binding sites for a signaling hairpin encoded with two C-rich templates of green- and red-AgNCs clustering. The identical ligation of signal probe with three arms of TWJDC liberates its locked stem, enabling the separate growth of red-clusters in three branches. As demonstrated, three clusters of red-AgNCs possess advantageous self-enhancing fluorescent performance relative to single or two cluster(s), good biocompatibility and low cytotoxicity. Utilizing the bicolor AgNCs as dual-emitters with reversely changed emission intensity, we developed an innovative ratiometric strategy displaying sensitively linear dose-dependence on variable T* down to 1.9 pM, which can afford a promising platform for biosensing, bioanalysis, cell imaging, or even clinical theranostics.

Innovative ratiometric optical strategy: Nonconjugated polymer dots based fluorescence-scattering dual signal output for sensing mercury ions

A new ratiometric platform was developed for sensing Hg2+, which combined fluorescence and scattering simultaneously. This ratiometric strategy reflected superiorities over conventional methods, since the two independent signals at irrelevant categories meet the requirements of sufficient wavelength separation, stimulation under one excitation, and collection on single instrument. Herein, nonconjugated polymer dots (N-PDs) were served as the recognition unit for Hg2+ with turn-off fluorescence and turn-on scattering. Additionally, two signal collection tactics were proposed to achieve fluorescence and scattering in a window: one was to record down-conversion fluorescence and second-order scattering spectra (FL@SOS), and the other was to gather the fluorescence excited by second-order diffraction light and first-order scattering (SODL-FL@FOS). This ratiometric sensor exhibited outstanding performance toward Hg2+ in the range of 0.1–50 μM with the detection limit of 27 nM. By contrast, the present proposal provided a more ingenious and scalable way to construct ratiometric sensor than traditional approach.

Enhancing Dye‐Triplet‐Sensitized Upconversion Emission Through the Heavy‐Atom Effect in CsLu2F7:Yb/Er Nanoprobes

AbstractLanthanide (Ln3+)‐doped upconversion (UC) nanoprobes, which have drawn extensive attention for various bioapplications, usually suffer from small absorption cross‐sections and weak luminescence intensity of Ln3+ ions. Herein, we report the controlled synthesis of a new class of Ln3+‐doped UC nanoprobes based on CsLu2F7:Yb/Er nanocrystals (NCs), which can effectively increase the intersystem crossing (ISC) efficiency from singlet excited state to triplet excited state of IR808 up to 99.3 % through the heavy atom effect. By virtue of the efficient triplet sensitization of IR808, the optimal UC luminescence (UCL) intensity of IR808‐modified CsLu2F7:Yb/Er NCs is enhanced by 1309 times upon excitation at 808 nm. Benefiting from the intense dye‐triplet‐sensitized UCL, the nanoprobes are demonstrated for sensitive assay of extracellular and intracellular hypochlorite with an 808‐nm/980‐nm dual excited ratiometric strategy.

Enhancing Dye-Triplet-Sensitized Upconversion Emission Through the Heavy-Atom Effect in CsLu2 F7 :Yb/Er Nanoprobes.

Lanthanide (Ln3+ )-doped upconversion (UC) nanoprobes, which have drawn extensive attention for various bioapplications, usually suffer from small absorption cross-sections and weak luminescence intensity of Ln3+ ions. Herein, we report the controlled synthesis of a new class of Ln3+ -doped UC nanoprobes based on CsLu2 F7 :Yb/Er nanocrystals (NCs), which can effectively increase the intersystem crossing (ISC) efficiency from singlet excited state to triplet excited state of IR808 up to 99.3 % through the heavy atom effect. By virtue of the efficient triplet sensitization of IR808, the optimal UC luminescence (UCL) intensity of IR808-modified CsLu2 F7 :Yb/Er NCs is enhanced by 1309 times upon excitation at 808 nm. Benefiting from the intense dye-triplet-sensitized UCL, the nanoprobes are demonstrated for sensitive assay of extracellular and intracellular hypochlorite with an 808-nm/980-nm dual excited ratiometric strategy.

Label-free ratiometric homogeneous electrochemical aptasensor based on hybridization chain reaction for facile and rapid detection of aflatoxin B1 in cereal crops

Aflatoxin B1 (AFB1) contamination has raised global concerns in agricultural and food industry; thus, sensitive, accurate and rapid AFB1 sensors are essential in many circumstances. Herein, we developed a label-free and immobilization-free ratiometric homogeneous electrochemical aptasensor based on hybridization chain reaction (HCR) for facile and rapid determination of AFB1. Methylene blue (MB) and ferrocene (Fc) were used as label-free probes to produce a response signal (IMB) and a reference signal (IFc) in solution phase, respectively. The ratio of IMB/IFc was used as a yardstick to quantify AFB1. HCR was exploited to enlarge the intensity of IMB as well as ratiometric signal. By combining label-free homogeneous assay and ratiometric strategy, the resulting aptasensor offered sensitive, rapid, and reliable determinations of AFB1 with a detection limit of 38.8 pg mL−1. The aptasensor was then used to determine AFB1 in cereal samples with comparable reliability as HPLC-MS.

Cu-THQ metal-organic frameworks: A kind of new inner reference for the reliable detection of dopamine base on ratiometric electrochemical sensing

Ratiometric electrochemical sensing provides a new perspective for the accurate and reliable detection of different analytes since inner reference signal can effectively eliminate the internal and external interferences. There is still a challenge to explore new inner references for the development of ratiometric electrochemical sensing. Herein, a kind of redox-active metal-organic frameworks (Cu-THQ MOFs) was synthesized and characterized by scanning electron microscope, transmission electron microscope, X-ray powder diffractometer, and X-ray photoelectron spectroscopy techniques. Cu-THQ MOFs were composited with carboxylic group functionalized multi-walled carbon nantubes (MWCNTs-COOH) to construct a sensitive electrochemical sensor. The modified Cu-THQ MOFs not only showed good catalytic active to dopamine (DA), but also displayed the constant peak current with the increase of concentration of DA. And a separated oxidation peak potential of about 270 mV between DA and Cu-THQ MOFs can be observed. Accordingly, a current ratio (iDA/iMOFs) was employed as readout signal for DA detection with a linear range and limit of detection of 3.0 × 10−7– 4.0 × 10−5 mol/L and 2.6 × 10−8 mol/L, respectively. Compared with single signal of iDA, ratiometric strategy exhibited high accuracy and reliability for DA determination. The present ratiometric sensing strategy using MOFs as the inner reference may provide a new choice for future development of electrochemical sensors for other analytes reliable determination.

Sensitive detection of trace 4-methylimidazole utilizing a derivatization reaction-based ratiometric surface-enhanced Raman scattering platform

Herein, a facile and fast surface-enhanced Raman scattering (SERS) method with ratiometric strategy was developed for detection of 4-methylimidazole (4-MI). Via a chemical derivatization reaction with 3-amino-5-mercapto-1,2,4-triazole (AMTA) diazonium salts, 4-MI could be converted to SERS-sensitive species. The SERS intensity ratio between the peaks at 1243 cm−1 and 1110 cm−1 (I1243/I1110) was used for the quantification of 4-MI. In addition, the method sensitivity was further improved by the aggregation of beta-cyclodextrin-modified Ag nanoparticles (beta-CD-AgNPs). Under the optimal conditions, the limit of detection (LOD) and the limit of quantification (LOQ) for 4-MI were 1.7 nM (S/N = 3) and 5.7 nM (S/N = 10), respectively. The relative standard deviation (RSD) for 0.5 μM 4-MI was 8.2% (n = 20). This method was successfully used for the determination of 4-MI in cola samples with recoveries ranging from 92% to 106%. The present method is convenient, sensitive, selective, reliable and may have a promising application in determination of the compounds with an imidazole ring containing active hydrogen atoms.

LAMP-H+-responsive electrochemical ratiometric biosensor with minimized background signal for highly sensitive assay of specific short-stranded DNA

Herein, the sequence-specific short-stranded biomarker DNA (hDNA, 21-nt) is acted as targeting out-primer to implement the loop-mediated isothermal amplification for releasing hydrogen ions (LAMP-H+). Using LAMP-H+ as signaling transducer, we report a highly sensitive electrochemical ratiometric biosensor for hDNA with minimized background signal, which is achieved via magnetic separation using AuNPs-modified Fe3O4 (Au@Fe3O4) as micro-reactor. In Au@Fe3O4, a double-stranded complex of a pH-responsible strand (I*) and a substrate strand (S*) is bound via Au–N bonds, where the treatment with LAMP-H+ leads to I* folding into i-motif conformation and S* dehybridization. The S* further hybridizes a catalytic strand (C*) to assemble Mg2+-DNAzymes that are cleaved by Mg2+, releasing C* for repeated formation and robust nicking of Mg2+-DNAzymes. The resultant output fuel strands (F*) are introduced in a modified electrode to drive the strand displacement of two hairpins individually labeled with two electron mediators. Through F*-mediated recycled amplification, the ratio of their electrochemical currents changed in opposite is highly sensitive to the varied hDNA down to 2.1 fM. By integrating LAMP-H+-stimulated i-motif switching with Mg2+-DNAzyme cleavage, this logic transduction of LAMP-H+(i-motif/Mg2+-DNAzyme)F* efficiently minimizes the inherent background of traditional LAMP-based assays. Resultantly, our electrochemical ratiometric strategy would be applicable to diverse short-stranded DNAs or even RNAs as targeting primers of LAMP.

A dual-signaling electrochemical ratiometric strategy combining “signal-off” and “signal-on” approaches for detection of MicroRNAs

A dual-signaling electrochemical ratio metric strategy was developed for detection microRNA-18a based on the duplex-specific nuclease-assisted target recycling and electrochemical atom transfer radical polymerization signal amplification. In the presence of target microRNA, RNA/DNA duplexes are formed, which become the substrate of the duplex-specific nuclease-assisted target recycling. Hence only the DNA strand is cleaved by duplex-specific nuclease enzyme, resulting in the throw away of methylene blue (MB) from the electrode (signal off) accompanied by releasing of target microRNA, which can be recycled in the next hybridization. The remaining piece of capture DNAs on the electrode surface hybridize with the Azide labeled-signal DNAs. “Click reactions” were carried out between 3-Butynyl-2-bromoisobutyrate and Azide to initiate the electrochemical atom transfer radical polymerization reaction. This process could bring a great number of ferrocenylmethyl methacrylate (FMMA) on the surface of electrode (signal on). The IFMMA/IMB value was proportionate to the microRNA-18a concentration and measured by square wave voltammetry.The promising potential of the proposed biosensor in clinical analyses was exhibited by its remarkable features such as strong performance, high specificity, agreeable storage stability, and notable selectivity in real sample evaluation with no pretreatment or amplification. Finally, our biosensing method offers such an application to be used for the early clinical diagnosis of Pancreatic Cancer.

Antibody-Responsive Ratiometric Fluorescence Biosensing of Biemissive Silver Nanoclusters Wrapped in Switchable DNA Tweezers.

Exploring the ratiometric fluorescence biosensing of DNA-templated biemissive silver nanoclusters (AgNCs) is significant in bioanalysis, yet the design of a stimuli-responsive DNA device is a challenge. Herein, using the anti-digoxin antibody (anti-Dig) with two identical binding sites as a model, a tweezer-like DNA architecture is assembled to populate fluorescent green- and red-AgNCs (g-AgNCs and r-AgNCs), aiming to produce a ratio signal via specific recognition of anti-Dig with two haptens (DigH). To this end, four DNA probes are programmed, including a reporter strand (RS) dually ended with a g-/r-AgNC template sequence, an enhancer strand (ES) tethering two same G-rich tails (G18), a capture strand (CS) labeled with DigH at two ends, and a help strand (HS). Initially, both g-AgNCs and r-AgNCs wrapped in the intact RS are nonfluorescent, whereas the base pairing between RS, ES, CS, and HS resulted in the construction of DNA mechanical tweezers with two symmetric arms hinged by a rigid "fulcrum", in which g-AgNCs are lighted up due to G18 proximity ("green-on"), and r-AgNCs away from G18 are still dark ("red-off"). When two DigHs in proximity recognize and bind anti-Dig, the conformation switch of these tweezers resultantly occurs, taking g-AgNCs away from G18 for "green-off" and bringing r-AgNCs close to G18 for "red-on". As such, the ratiometric fluorescence of r-AgNCs versus g-AgNCs is generated in response to anti-Dig, achieving reliable quantization with a limit of detection at the picomolar level. Based on the fast stimulated switch of unique DNA tweezers, our ratiometric strategy of dual-emitting AgNCs would provide a new avenue for a variety of bioassays.