Fluorescent Platform Research Articles

Fluorescence lifetime multiplexing with fluorogen activating protein FAST variants

In this paper, we propose a fluorescence-lifetime imaging microscopy (FLIM) multiplexing system based on the fluorogen-activating protein FAST. This genetically encoded fluorescent labeling platform employs FAST mutants that activate the same fluorogen but provide different fluorescence lifetimes for each specific protein-dye pair. All the proposed probes with varying lifetimes possess nearly identical and the smallest-in-class size, along with quite similar steady-state optical properties. In live mammalian cells, we target these chemogenetic tags to two intracellular structures simultaneously, where their fluorescence signals are clearly distinguished by FLIM. Due to the unique structure of certain fluorogens under study, their complexes with FAST mutants display a monophasic fluorescence decay, which may facilitate enhanced multiplexing efficiency by reducing signal cross-talks and providing optimal prerequisites for signal separation upon co-localized and/or spatially overlapped labeling.

Label-free fluorescence platform based on SiO2-coated CdTeS quantum dots for trace analysis of Ag+ in environmental water

In this study, a core-shell structural nano-composite material, namely CdTeS@SiO2, is synthesized by a simple silanization of Te-doped CdS quantum dots (CdTeS QDs). Through SiO2 capping, CdTeS QDs not only improve the fluorescence performance effectively, but also greatly enhance the anti-interference ability in the environment. Based on its excellent optical properties, a novel fluorescence sensor is constructed for the ultramicro detection of Ag+. The fluorescence of CdTeS@SiO2 is strongly quenched in the presence of Ag+ and shows good linearity in the range of 0.005–5.0 μmol L−1 with a detection limit as low as 1.6 nmol L−1. This is mainly due to its unique quenching mechanism: Ag+ destroys the spherical structure of SiO2 and promotes the formation of non-radiative electron-hole pairs through electron transfer, leading to fluorescence quenching. At the same time, it competes with Cd for Te, S and MPA on the CdTeS surface, forming Ag–Te, Ag–S and Ag–MPA complexes attached to the CdTeS surface leading to wavelength red-shift. The feasibility of the proposed sensor is demonstrated through spiking experiments, which confirmed the potential value of the constructed fluorescence probe for real-world applications in detecting Ag+ in environmental water.

A smartphone-integrated ratiometric fluorescent platform based on europium (III)-functionalized metal-organic frameworks for quantitative and visual detection of tetracycline

Food safety and environmental pollution caused by tetracycline antibiotics (TCs) are of great concern for public health worldwide. To realize on-site, real-time, ultrasensitive visual detection of tetracycline is an urgent need. Herein, a ratiometric fluorescent probe has been constructed by grafting Cit-Eu3+ on the amino-silanes-modified metal–organic framework for the detection of tetracycline. Under exciting at 330 nm,the red emission at 617 nm of Eu3+ was significantly enhanced by the antenna effect upon adding tetracycline, while the intrinsic fluorescence quenching at 425 nm of the probe ascribes to the inner filter effect, resulting in a reversible signal response. Under optimum conditions, the probe exhibits a visualized fluorescence evolution from blue to red with a linear range of 0.1–60 μM, and a limit of detection as low as 24.6 nM. Furthermore, a test strip was further fabricated by dropping NH2-MIL(Al)@SiO2-Cit-Eu3+ on filter paper, which can be used for on-site of tetracycline conveniently by using a smartphone. Moreover, the proposed probe has also been successfully applied for TCs detection in spiked honey and tap water sample with satisfactory recoveries in the range from 91.21–102.97 %, demonstrating the potential real application performance. This the smart sensing platform lays a foundation for the rapid screening of TCs in food and environmental matrices.

BPGQD/g-C3N4 nanocomposites as sensitive and selective platform for fluorescence and RGB based detection of vitamin B12

Here, a brand-new fluorescence BPGQD/g-C3N4 nanocomposite for measuring Vitamin B12 has been created. The technique is based on the interaction between Vitamin B12 and graphitic nitride nanosheets modified with graphene quantum dots co-doped with boron and phosphorus. The developed nanocomposites have been characterized by using various techniques such as HR-TEM, SEM, EDS, XRD, FT-IR, XPS, Photoluminescence and UV–Vis absorption spectroscopy. Different excitation wavelengths were used, with a 320 nm excitation wavelength and a 416 nm emission wavelength chosen for fluorometric detection. It examined how several factors, including the sample solution's pH, the length of the incubation period, and the impact of NaCl concentration, affected the intensity of the fluorescence. A linear standardization curve was validated in the Vitamin B12 range of 0–60 nM under the authorized conditions. The limit of detection (LOD) and limit of quantitation (LOQ) were 9.61 nM and 29.12 nM respectively. Using newly created nanocomposites, an RGB-based sensor for Vitamin B12 detection was also constructed. Using this probe, it was possible to determine the amount of Vitamin B12 in human urine samples with respectable outcomes and satisfactory recoveries.

Reactivity-Tunable Fluorescent Platform for Selective and Biocompatible Modification of Cysteine or Lysine.

Chemoselective modification of specific residues within a given protein poses a significant challenge, as the microenvironment of amino acid residues in proteins is variable. Developing a universal molecular platform with tunable chemical warheads can provide powerful tools for precisely labeling specific amino acids in proteins. Cysteine and lysine are hot targets for chemoselective modification, but current cysteine/lysine-selective warheads face challenges due to cross-reactivity and unstable reaction products. In this study, a versatile fluorescent platform is developed for highly selective modification of cysteine/lysine under biocompatible conditions. Chloro- or phenoxy-substituted NBSe derivatives effectively labeled cysteine residues in the cellular proteome with high specificity. This finding also led to the development of phenoxy-NBSe phototheragnostic for the diagnosis and activatable photodynamic therapy of GSH-overexpressed cancer cells. Conversely, alkoxy-NBSe derivatives are engineered to selectively react with lysine residues in the cellular environment, exhibiting excellent anti-interfering ability against thiols. Leveraging a proximity-driven approach, alkoxy-NBSe probes are successfully designed to demonstrate their utility in bioimaging of lysine deacetylase activity. This study also achieves integrating a small photosensitizer into lysine residues of proteins in a regioselective manner, achieving photoablation of cancer cells activated by overexpressed proteins.

Sulfate radical-dominated colorimetric sensing platform for ultrafast and portable monitoring of tetracycline in environment

Tetracycline (TC) is one of the most frequently detected antibiotics in environmental. Monitoring the concentration of TC is essential for understanding environmental fate of antibiotics. In this work, bimetallic N-doped carbon catalysts of M/Co@NC (M= Ni, Mn, Cu, Zn, Fe) were rationally synthesized via molten salt-assisted pyrolysis. The distinctive synergistic interplay between Cu and Co endowed Cu/Co@NC superior activation capacity for peroxymonosulphate (PMS). Inspired by the advantages of sulfate radical (SO4•−), an original colorimetric sensing platform was developed by using PMS as oxidant and rhodamine B (RhB) as chromogenic agents. Notably, this platform achieved an ultrafast visual reaction within 60 s and exhibited broad pH applicability under pH 2–9. Then, leveraging a competitive inhibition mechanism, dual-mode colorimetric and fluorescent sensing platforms were established for TC detection under neutral conditions. The fluorescent platform exhibited a reliable liner range from 0.001 to 15 μg/mL with limit of detection of 2 ng/mL. Excellent selectivity was found for TC even among different antibiotics. Theoretical calculations elucidated that the superior selectivity arises from a higher electrophilic attack (f-) of TC. Based on these finding, a smartphone-assisted test strip was developed and successfully deployed for the rapid monitoring of TC in practical water samples. This work not only synthesized an efficient PMS activator, but also proposed a novel visual sensing mode with ultrafast detection times, wide pH applicability and portable operation.

A handheld fluorescent lateral flow immunoassay platform for highly sensitive point-of-care detection of methamphetamine and tramadol

The escalating issue of drug abuse poses a significant threat to public health and societal stability worldwide. An on-site drug detection platform is vital for combating drug abuse and trafficking, as it eliminates the need for additional tools, extensive processes, or specialized training. Therefore, it is imperative to develop a fast, sensitive, non-invasive, and reliable multiplex drug testing platform. In this study, we have presented a silica core@dual quantum dot-shell nanocomposite (SI/DQD)-based fluorescent lateral flow immunoassay (LFIA) platform for the highly sensitive and simultaneous point-of-care (POC) detection of methamphetamine (MET) and tramadol (TR). A 3D-printed attachment was designed to integrate optical and electrical components, facilitating the miniaturization of the instrument and reducing both cost and complexity. The device's advanced hardware and effective fluorescence extraction algorithm with waveform reconstruction enable swift, automatic noise reduction and data analysis. SI/DQD nanocomposites were utilized as fluorescent nanotags in the LFIA strips due to their outstanding luminous efficiency and robustness. This LFIA platform achieves impressive detection limits (LODs) of 0.11 ng mL−1 for MET and 0.017 ng mL−1 for TR. The method has also successfully detected MET and TR in complex biological samples, demonstrating its practical application capabilities. The proposed fluorescent LFIA platform, based on SI/DQD technology, holds significant promise for the swift and accurate POC detection of these substances. Its affordability, compact size, and excellent analytical performance make it suitable for on-site drug testing, including at borders and roadside checks, and open up new possibilities for the design and implementation of drug testing methods.

Optofluidic photonic crystal fiber platform for sensitive and reliable fluorescence based biosensing.

Biosensing plays a pivotal role in various scientific domains, offering significant contributions to medical diagnostics, environmental monitoring, and biotechnology. Fluorescence biosensing relies on the fluorescence emission from labelled biomolecules to enable sensitive and selective identification and quantification of specific biological targets in various samples. Photonic crystal fibers (PCFs) have led to the development of optofluidic fibers enabling efficient light-liquid interaction within small liquid volume. Herein, we present the development of a user-friendly optofluidic-fiber platform with simple hardware requirements for sensitive and reliable fluorescence biosensing with high measurement repeatability. We demonstrate a sensitivity improvement of the fluorescence emission up to 17 times compared to standard cuvette measurement, with a limit of detection of Cy5 fluorophore as low as 100 pM. The improvement in measurement repeatability is exploited for detecting haptoglobin protein, a relevant biomarker to diagnose several diseases, by using commercially available Cy5 labelled antibodies. The study aims to showcase an optofluidic platform leveraging the benefits provided by optofluidic fibers, which encompass easy light injection, robustness, and high sensitivity.

First-in-man study of the PSMA Minibody IR800-IAB2M for molecularly targeted intraoperative fluorescence guidance during radical prostatectomy

PurposeProstate-specific membrane antigen (PSMA) is increasingly used to image prostate cancer in clinical practice. We sought to develop and test a humanised PSMA minibody IAB2M conjugated to the fluorophore IRDye 800CW-NHS ester in men undergoing robot-assisted laparoscopic radical prostatectomy (RARP) to image prostate cancer cells during surgery.MethodsThe minibody was evaluated pre-clinically using PSMA positive/negative xenograft models, following which 23 men undergoing RARP between 2018 and 2020 received between 2.5 mg and 20 mg of IR800-IAB2M intravenously, at intervals between 24 h and 17 days prior to surgery. At every step of the procedure, the prostate, pelvic lymph node chains and extra-prostatic surrounding tissue were imaged with a dual Near-infrared (NIR) and white light optical platform for fluorescence in vivo and ex vivo. Histopathological evaluation of intraoperative and postoperative microscopic fluorescence imaging was undertaken for verification.ResultsTwenty-three patients were evaluated to optimise both the dose of the reagent and the interval between injection and surgery and secure the best possible specificity of fluorescence images. Six cases are presented in detail as exemplars. Overall sensitivity and specificity in detecting non-lymph-node extra-prostatic cancer tissue were 100% and 65%, and 64% and 64% respectively for lymph node positivity. There were no side-effects associated with administration of the reagent.ConclusionIntraoperative imaging of prostate cancer tissue is feasible and safe using IR800-IAB2M. Further evaluation is underway to assess the benefit of using the technique in improving completion of surgical excision during RARP.RegistrationISCRCTN10046036: https://www.isrctn.com/ISRCTN10046036.

Rapid detection of the irinotecan-related UGT1A1 & 5-fluorouracil related DPYD polymorphism by asymmetric polymerase chain reaction melting curve analysis

BackgroundDetermination of DPYD and UGT1A1 polymorphisms prior to 5-fluorouracil and irinotecan therapy is crucial for avoiding severe adverse drug effects. Hence, there is a pressing need for accurate and reliable genotyping methods for the most common DPYD and UGT1A1 polymorphisms. In this study, we introduce a novel polymerase chain reaction (PCR) melting curve analysis method for discriminating DPYD c.1236G > A, c.1679 T > G, c.2846A > T, IVS14 + 1G > A and UGT1A1*1, *28, *6 (G71R) genotypes. MethodsFollowing protocol optimization, this technique was employed to genotype 28 patients, recruited between March 2023 and October 2023, at the First Affiliated Hospital of Xiamen University. These patients included 20 with UGT1A1 *1/*1, 8 with UGT1A1 *1/*28, 4 with UGT1A1 *28/*28, 22 with UGT1A1*6 G/G, 6 with UGT1A1*6 G/A, 4 with UGT1A1*6 A/A, 27 with DPYD(c.1236) G/G, 3 with DPYD(c.1236) G/A, 2 with DPYD(c.1236) A/A, 27 with DPYD(c.1679) T/T, 2 with DPYD(c.1679) T/G, 3 with DPYD(c.1679) G/G, 28 with DPYD(c.2846A/T) A/A, 2 with DPYD(c.2846A/T) A/T, 2 with DPYD(c.2846A/T) T/T, 28 with DPYD(c.IVS14 + 1) G/G, 2 with DPYD(c.IVS14 + 1) G/G, and 2 with DPYD(c.IVS14 + 1) G/G, as well as 3 plasmid standards. Method accuracy was assessed by comparing results with those from Sanger sequencing or Multiplex quantitative PCR(qPCR). Intra- and inter-run precision of melting temperatures (Tms) were calculated to evaluate reliability, and sensitivity was assessed through limit of detection examination. ResultsThe new method accurately identified all genotypes and exhibited higher accuracy than Multiplex qPCR. Intra- and inter-run coefficients of variation for Tms were both ≤1.97 %, with standard deviations ≤0.95 °C. The limit of detection was 0.09 ng/μL of input genomic DNA. ConclusionOur developed PCR melting curve analysis offers accurate, reliable, rapid, simple, and cost-effective detection of DPYD and UGT1A1 polymorphisms. Its application can be easily extended to clinical laboratories equipped with a fluorescent PCR platform.

Early fetal sex determination using a fluorescent DNA nanosensing platform capable of simultaneous detection of SRY and DYS14 sequences in cell-free fetal DNA

Early fetal sex determination is of crucial importance in the management of prenatal diagnosis of X-linked genetic abnormalities and congenital adrenal hyperplasia. The development of an efficient and simple method for high-sensitivity, affordable, and rapid screening of cell-free fetal DNA (cffDNA) is crucial for fetal sex determination in early pregnancy. In this study, single- and dual-fluorophore DNA biosensors based on multi-walled carbon nanotubes (MWCNT) were fabricated for the individual and simultaneous detection of the SRY gene and DYS14 marker in cffDNA obtained from maternal plasma samples. This nanosensing platform is based on the immobilization of single-stranded DNA (ssDNA) probes, labeled with ROX or FAM fluorophores, on MWCNT, resulting in the quenching of fluorescence emission in the absence of the targets. Upon the addition of the complementary target DNA (ctDNA) to the hybridization reaction, the fluorescence emission of fluorophore-labeled probes was significantly recovered to 79.5 % for ROX-labeled probes (i.e. SRY-specific probes), 81.5 % for FAM-labeled probes (i.e. DYS14-specific probes), and 65.9 % for dual-fluorophore biosensor compared to the quenching mode. The limit of detection (LOD) for ROX, and FAM was determined to be 4.5 nM, and 7.6 nM, respectively. For dual-color probes, LOD was found to be 5.4 (ROX) and 9.2 nM (FAM). Finally, the clinical applicability of the proposed method was confirmed through the detection of both biomarkers in maternal plasma samples, suggesting that the proposed nanosensing platform may be useful for the early detection of fetal sex using cffDNA.

Development and clinical evaluation of a real-time multiple cross displacement amplification assay for rapid and sensitive detection of Mycobacterium tuberculosis

Molecular techniques of nucleic acid testing recommended by the World Health Organization (WHO) for the Mycobacterium tuberculosis (MTB) detection were considered to have the potential access to the accurate tuberculosis (TB) notifications. In this study, a new method, which coupled real-time (rt) fluorescence technique with multiple cross displacement amplification (MCDA), was developed for the rapid, sensitive and specific detection of MTB (termed MTB-rt-MCDA). According to the principle of the rt-MCDA test, a set of ten primers were designed for the MCDA reaction, of which one was engineered with a restrictive endonuclease recognition site, a fluorophore and a quencher for achieving the real-time fluorescence detection. MTB-rt-MCDA test was conducted under the optimized conditions (67 °C, 40 min) on the real-time fluorescence platform. The MTB-rt-MCDA assay accurately identified the MTB strains with no cross reaction with other bacteria. The lowest detectable genomic DNA concentration of the MTB-rt-MCDA assay was 25 fg/μl. We employed the genomic DNA templates extracted from sputum of clinical cases for validating the practical applicability of this assay, and the detection power of the MTB-rt-MCDA assay was comparable to that of the Xpert method and MCDA-based biosensor detection and superior to smear microscope method. The complete process of the MTB-rt-MCDA assay, including rapid extraction of DNA and rt-MCDA test, takes less than 1 h. In conclusion, the presented MTB-rt-MCDA assay provided an effective and simple option for the rapid screening of MTB infection.

A Portable Fluorescent Lateral Flow Immunoassay Platform for Rapid Detection of FluA.

The spread of the FluA virus poses significant public health concerns worldwide. Fluorescent lateral flow immunoassay (LFIA) test strips have emerged as vital tools for the early detection and monitoring of influenza infections. However, existing quantitative virus-detection methods, particularly those utilizing smartphone-based sensing platforms, encounter accessibility challenges in resource-limited areas and among the elderly population. Despite their advantages in speed and portability, these platforms often lack user-friendliness for these demographics, impeding their widespread utilization. To address these challenges, this study proposes leveraging the optical pick-up unit (OPU) sourced from commercial optical drives as a readily available fluorescence excitation module for the quantitative detection of antibodies labeled with quantum-dot fluorescent microspheres. Additionally, we utilize miniaturized and high-performance optical components and 3D-printed parts, along with a customized control system, to develop an affordable point-of-care testing (POCT) device. Within the system, a stepping motor scans the test strip from the T-line to the C-line, enabling the calculation of the fluorescence-intensity ratio between the two lines. This simple yet effective design facilitates rapid and straightforward field or at-home testing for FluA. The proposed prototype platform demonstrates promising performance, achieving a limit of detection (LOD) of 2.91 ng/mL, a total detection time of no more than 15 min, and dimensions of 151 mm × 11.2 mm × 10.8 mm3. We believe that the proposed approach holds great potential for improving access to an accurate influenza diagnosis.

Portable smartphone-based RecJf exonuclease-modulated enhanced ratiometric fluorescence bioplatform for rapid visual detection of As3+

Arsenite (As3+), a highly carcinogenic heavy metal ion and widely distributed in nature, can have serious health implications even with minimal exposure. Herein, a portable smartphone device-based ratiometric fluorescence platform was established for sensitive detection of As3+. The work relied on the use of metal-organic framework-tagged cDNA (PCN-224-cDNA), with high adsorption capability and fluorescence properties, as an internal reference to quench the fluorescence of FAM-anchored aptamer (FAM-Apt) via hybridization. In the presence of As3+, FAM-Apt specifically bound to As3+ leading to conformational changes, which detached from the PCN-224-cDNA surface. Interestingly, a smartphone-based readout equipment engineered using a 3D-printed hardware device administered the portable detection of As3+. The limit of detection (LOD) for the proposed ratiometric biosensor was calculated to be 0.021 ng/mL, significantly below WHO's safety threshold. Hence, it demonstrates significant potential for large-scale screening of As3+ residues in food and the environment.

A [Co5] cluster‐based organic framework as fluorescent detection platform toward quinolone antibiotics

The detection of quinolone antibiotics is an important research hotspot for protecting human health. Herein, a three‐dimensional metal–organic framework with the formula of {[Co5(TPT)2(BPDC)3(μ3‐O)2(H2O)4]·8MeCN·3DMF}n (NS‐3, TPT = tris(4‐pyridyl)‐1,3,5‐triazin, H2BPDC = 1,4‐benzenedicarboxylic acid) has been successfully prepared through solvothermal method. Single‐crystal X‐ray diffraction analysis revealed that three‐dimensional NS‐3 can be viewed as an eight‐connected sqc4 net with pentanuclear [Co5(μ3‐O)2]6+ cluster as metal nodes. Importantly, when norfloxacin, ofloxacin, and enrofloxacin were added in NS‐3 ethanol suspension, the luminescence intensity of NS‐3 at 333 nm was obviously decreased while new emission peaks clearly emerged, indicating that NS‐3 can be a potential rare fluorescent detection to recognize quinolone antibiotics in ethanol. Additionally, NS‐3 exhibits high sensitivity to norfloxacin, ofloxacin, and enrofloxacin with a limit of detection of 43, 34, and 139 nM, respectively, which is superior to most of the reported metal–organic frameworks.

Dual-ligand lanthanide metal-organic framework based ratiometric fluorescent platform for visual monitoring of aminoglycoside residues in food samples

Herein, a dual-emission Eu metal-organic framework (Eu-MOF) is prepared and used as the ratiometric fluorescence probe for ultrasensitive detection of aminoglycoside antibiotics (AGs). Due to the strong hydrogen bond interactions between AGs and Eu-MOF, the blue emission is enhanced while the red emission has little fluctuation in Eu-MOF with the addition of AGs, thus a good linear relationship with the logarithm of AGs concentrations from 0.001 to 100 μg/mL can be established for quantitative analysis. Good sensitivity with the detection limit of 0.33 ng/mL for apramycin, 0.32 ng/mL for amikacin and 0.30 ng/mL for kanamycin is achieved. The proposed assay demonstrates good selectivity and applicability for determination of AGs in real milk and honey samples. The Eu-MOF materials are further fabricated as fluorescent test papers for facile visual detection. The as-established ratio fluorescence platform offers a portable and economical way for rapid monitoring AGs residues in complex food samples.

A novel fluorescence platform for specific detection of tetracycline antibiotics based on [MQDA-Eu3+] system

Tetracycline antibiotics (TCs) are extensively used in clinical medicine, animal husbandry, and aquaculture because of their cost-effectiveness and high antibacterial efficacy. However, the presence of TCs residues in the environment poses risks to humans. In this study, an inner filter effect (IFE) fluorescent probe, 2,2′-(ethane-1,2-diylbis((2-((2-methylquinolin-8-yl)amino)-2-oxoethyl)azanediyl))diacetic acid (MQDA), was developed for the rapid detection of Eu3+ within 30 s. And its complex [MQDA–Eu3+] was successfully used for the detection of TCs. Upon coordination of a carboxyl of MQDA with Eu3+ to form a [MQDA–Eu3+] complex, the carboxyl served as an antenna ligand for the effective detection of Eu3+ to intensify the emission intensity of MQDA via “antenna effect”, the process was the energy absorbed by TCs via UV excitation was effectively transferred to Eu3+. Fluorescence quenching of the [MQDA–Eu3+] complex was caused by the IFE in multicolor fluorescence systems. The limits of detection of [MQDA–Eu3+] for oxytetracycline, chlorotetracycline hydrochloride, and tetracycline were 0.80, 0.93, and 1.7 μM in DMSO/HEPES (7:3, v/v, pH = 7.0), respectively. [MQDA–Eu3+] demonstrated sensitive detection of TCs in environmental and food samples with satisfactory recoveries and exhibited excellent imaging capabilities for TCs in living cells and zebrafish with low cytotoxicity. The proposed approach demonstrated considerable potential for the quantitative detection of TCs.

Fluorescence emission angular dependence on a nanostructured plasmonic grating

AbstractPlasmonic gratings provide an advantageous platform for fluorescence sensing due to their compatibility with functionalization techniques, imaging detection, and the potential for signal enhancement. Among traditional fluorescence detection methods, microscopes are commonly used tools. The interaction between dye fluorescence processes and the plasmonic modes of the grating strongly depends on the measurement configuration and is influenced by the dispersion and spectral characteristics of the plasmonic modes. In our study, we investigated the angular behavior of the fluorescence emission from ATTO700 dye by varying the collection angle using a standard optical microscope coupled with a spectrometer. Our results show a clear dependence of fluorescence emission in terms of spectral shape on the collection angle that can be attributed to plasmonic mode dispersion.

Programmable Entropy-Driven Circuit-Cascaded Self-Feedback DNAzyme Network for Ultra-Sensitive Fluorescence and Photoelectrochemical Dual-Mode Biosensing.

Inspired by natural DNA networks, programmable artificial DNA networks have become an attractive tool for developing high-performance biosensors. However, there is still a lot of room for expansion in terms of sensitivity, atom economy, and result self-validation for current microRNA sensors. In this protocol, miRNA-122 as a target model, an ultrasensitive fluorescence (FL) and photoelectrochemical (PEC) dual-mode biosensing platform is developed using a programmable entropy-driven circuit (EDC) cascaded self-feedback DNAzyme network. The well-designed EDC realizes full utilization of the DNA strands and improves the atomic economy of the signal amplification system. The unique and rational design of the double-CdSe quantum-dot-released EDC substrate and the cascaded self-feedback DNAzyme amplification network significantly avoids high background signals and enhances sensitivity and specificity. Also, the enzyme-free, programmable EDC cascaded DNAzyme network effectively avoids the risk of signal leakage and enhances the accuracy of the sensor. Moreover, the introduction of superparamagnetic Fe3O4@SiO2-cDNA accelerates the rapid extraction of E2-CdSe QDs and E3-CdSe QDs, which greatly improves the timeliness of sensor signal reading. In addition to the strengths of linear range (6 orders of magnitude) and stability, the biosensor design with dual signal reading makes the test results self-confirming.

A fluorescence-electrochemical dual-mode aptasensor based on novel DNA-dependent PBNFs@PtPd for highly selective and sensitive detection of procymidone through hybridization chain reaction

Herein, a study for the first application of a hybridization chain reaction, a 1,8-naphthalimides-DNA (NDs) intercalator, and DNA-dependent Prussian blue nanoflowers@PtPd materials (PBNFs@PtPd) in the development of a fluorescence-electrochemical (FL-EC) aptasensor. This construction establishes an efficient sensing platform for the detection of procymidone (PCM). In the context of the described experiment, dual-mode detection is achieved through the generation of FL signals by an aptamer labeled with a Cy5 moiety and the formation of DPV signals by the modification of a thionine-appended 1,8-naphthalimide (Thi-NDs). In the presence of PCM, specific recognition occurs, followed by the utilization of magnetic separation technology to release DNA1 (S1) and aptamer-Cy5 (Apt-Cy5), subsequently introducing them onto both fluorescence and EC platforms. The presence of S1 effectively activates hybridization chain reaction (HCR) for the electrode surface, thereby significantly increasing the binding sites for Thi-NDs and consequently greatly amplifying the response signal of differential pulse voltammetry (DPV). The developed FL-EC dual-mode sensing platform demonstrates high sensitivity in the detection of PCM, with the detection limits of 0.173 μg·ml−1 (within the detection range of 500 pg·ml−1 to 500 ng·ml−1) and 0.074 ng·ml−1 (within the detection range of 100 pg·ml−1 to 100 ng·ml−1), respectively. The designed dual-mode sensor exhibits notable characteristics, including high selectivity, reproducibility, synergy, and reliable monitoring/capability for PCM in real samples.