A signal-on fluorescent aptasensor based on gold nanoparticles for kanamycin detection

This study describes the development, verification and practical application of an aptasensor for the fluorometric detection of kanamycin. Using the nucleic acid aptamer with FAM fluorescent group as the conjugate, using gold nanoparticles as the fluorescence dynamic quenching source, a fluorescence sensor was fabricated through the signal-on method for the micro-detection of kanamycin. The nucleic acid chimera is connected to the fluorophore, and the gold nanoparticles are used as the fluorescence dynamic quenching source under actual conditions. The detection limit of kanamycin is 0.1 pM, and the detection range is 0.1 pM to 0.1 μM. This biosensor works satisfactorily in complex samples with no impurities, which gives this method an obvious advantage over other analytical methods. In addition, the mechanism of action between gold nanoparticles/FAM–aptamer/kanamycin is discussed and studied in depth here. It provides a more thorough analysis and more application possibilities for fluorescence-aptamer biosensing.

This study describes the development, verification and practical application of an aptasensor for the fluorometric detection of kanamycin.     

Immunoassay-aptasensor for the determination of tumor-derived exosomes based on the combination of magnetic nanoparticles and hybridization chain reaction

The detection of tumor-related exosomes is of great significance. In this work, a fluorescence aptasensor was designed for the determination of tumor-related exosomes based on the capture of magnetic nanoparticles (MNPs) and specific recognition of an aptamer. MNPs were used as substrates to capture the exosomes by modifying the CD63 antibody on the MNP surface. Probe 1 consists of PDL-1 aptamer sequence and a section of other sequences. PDL-1 expression was observed on the surface of exosomes; the aptamer of PDL-1 could combine with PDL-1 with high affinity. Thus, the immunoassay-type compounds of “MNPs–exosomes–probe 1” were formed. The other section of probe 1 triggered the HCR with probe 2 and probe 3 and formed the super-long dsDNA. The addition of GelRed resulted in the generation of an amplified fluorescence signal. The proposed design demonstrated a good linearity with the exosome concentration ranging from 300 to 10⁷ particles per mL and with a low detection limit of 100 particles per mL. This aptasensor also exhibited high specificity for tumor-related exosomes, and was successfully applied in biological samples.

The detection of tumor-related exosomes is of great significance.     

Small-molecule fluorescent probes and their design

Small-molecule fluorescent probes have become powerful tools for using light to advance the study of cell biology, discover new drugs, detect environmental contaminants, and further the detection of cancer. These applications correlate with the expansion of the fluorescent probe research community – small in the late 20^th century, now a collection of more than a hundred research groups world-wide. This expansion required the entry of adventurous scientists from many other fields. This tutorial review introduces some important concepts related to fluorescent probe development. It is hoped that it will facilitate further expansion of the field by demystifying it.

Small-molecule fluorescent probes allow light to be used as a tool to advance the study of biology, discover new drugs, and further the detection of cancer. This tutorial review introduces important concepts related to fluorescent probe development.     

Rapid detection of staphylococcal enterotoxin B in milk samples based on fluorescence hybridization chain reaction amplification

A rapid, simple, and sensitive method has been developed to detect staphylococcal enterotoxin B (SEB). To establish the hybridization chain reaction-based aptasensor, we described the new probes of two hairpins (H1 and H2), which were first designed based on the partial complementary sequence of the SEB aptamer (cDNA). The H1 labeled with a fluorophore and a quencher can act as a molecular fluorescence “switch”. Hence, in the presence of SEB, the aptamer binds SEB, while the unbound cDNA triggers HCR to carry out the cyclic hybridization of H1 and H2 so as to turn “ON” the fluorescence through forming long nicked DNA. By using this new strategy, SEB can be sensitively detected within the range of 3.13 ng mL⁻¹ to 100 ng mL⁻¹ with a detection limit of 0.33 ng mL⁻¹ (S/N = 3). Furthermore, the developed method could facilitate the detection of SEB effectively in milk samples.

A new competitive aptasensor combined with HCR was developed for SEB detection.     

Synthesis and application of coumarin fluorescence probes

In recent years, the research on fluorescent probes has developed rapidly. Coumarin fluorescent probes have also been one of the hot topics in recent years. For the synthesis and application of coumarin fluorescent probes, great progress has been made. Coumarin fluorescent probes have become more and more widely used in biochemistry, environmental protection, and disease prevention, and have broad prospects. This review introduces the three main light emitting mechanisms (PET, ICT, FRET) of fluorescent probes, and enumerates some probes based on this light emitting mechanism. In terms of the synthesis of coumarin fluorescent probes, the existing substituents on the core of coumarin compounds were modified. Based on the positions of the modified substituents, some of the fluorescent probes reported in the past ten years are listed. Most of the fluorescent probes are formed by modifying the 3 and 7 position substituents on the mother nucleus, and the 4 and 8 position substituents are relatively less modified. In terms of probe applications, the detection and application of coumarin fluorescent probes for Cu²⁺, Hg²⁺, Mg²⁺, Zn²⁺, pH, environmental polarity, and active oxygen and sulfide in the past ten years are mainly introduced.

In recent years, the research on fluorescent probes has developed rapidly.     

One-step synthesis of M13 phage-based nanoparticles and their fluorescence properties

Fluorescent carbon nanoparticles have been gaining more attention in recent years for their excellent fluorescence properties and simple synthesis routes. Different carbon sources have been reported for fluorescent carbon nanoparticle synthesis but the use of virus particles as a carbon source is scarce. Herein, we report the utilization of M13 bacteriophage particles as the carbon source to synthesize phage-based nanoparticles through facile, one-step microwave heating. M13 bacteriophage is a nanosized filamentous virus particle with a single-stranded DNA genome encapsulated by a large number of coat proteins. These amino acid rich building blocks provide a substantial amount of carbon source for the synthesis of fluorescent nanoparticles. The resulting nanoparticles from M13 bacteriophage showed good water solubility and exhibited bright blue luminescence. The selectivity and sensitivity of the phage-based nanoparticles towards Fe(iii) ions showed a quenching effect with a linear correlation and a detection limit of 8.0 μM. This process highlights the potential application of virus particles as a source for the synthesis of fluorescent carbon nanoparticles and the sensing application.

M13 bacteriophage is an attractive alternative source for fluorescent nanoparticle synthesis.     

Near-infrared turn-on fluorescent probe for discriminative detection of Cys and application in in vivo imaging

Near-infrared (NIR) fluorescent probes are widely employed in biological detection because of their lower damage to biological samples, low background interference, and high signal-to-noise ratio. Herein, a highly water-soluble NIR probe (NIRHA) based on a hemicyanine skeleton and bearing an acrylate moiety was synthesized. The probe showed high selectivity toward cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH). The probe also had low cytotoxicity and was successfully applied in HeLa cells and mouse experiments. Results of bioimaging experiments indicated that the probe was effective for visualizing endogenous Cys in vitro and in vivo.

Near-infrared (NIR) fluorescent probes are widely employed in biological detection because of their lower damage to biological samples, low background interference, and high signal-to-noise ratio.     

Reporter-recruiting bifunctional aptasensor for bioluminescent analytical assays

We report a novel bioluminescent aptasensor, which consists of 2′-F-RNA aptamer modules joined into a bi-specific aptamer construct. One aptamer module binds the analyte, then after structural rearrangement the second module recruits non-covalently Ca²⁺-dependent photoprotein obelin from the solution, thus providing a bioluminescent signal. This concept allows using free protein as a reporter, which brings such advantages as no need for aptamer–protein conjugation, a possibility of thermal re-folding of aptamer component with no harm to a protein, and simpler detection protocol. We developed the new 2′-F-RNA aptamer for obelin, and proposed the strategy for engineering structure-switching bi-modular aptamer constructs which bind the analyte and the obelin in a sequential manner. With the use of hemoglobin as a model analyte, we showed the feasibility of utilizing the aptasensor in a fast and straightforward bioluminescent microplate assay. With a proper design of a secondary structure, this strategy of aptasensor engineering might be further extended to bi-specific aptamer-based bioluminescent sensors for other analytes of interest.

A novel structure-switching bioluminescent 2′-F-RNA aptasensor consists of analyte-binding and obelin-recruiting modules, joined into a bi-specific aptamer construct.     

Fluorescent probes for the detection of magnesium ions (Mg2+): from design to application

Magnesium ions (Mg²⁺) play essential roles in various physiological and pathological processes, its abnormal homeostasis in cells is related to many diseases, such as diabetes, neuromuscular disorders, hypertension and other cardiovascular disorders. Investigation on the regulation of magnesium in cellular processes has attracted considerable interest in the past several decades. Among those reported strategies, fluorescent imaging technology has become a powerful and cost-effective tool for the real-time monitoring of magnesium distribution, uptake and trafficking, due to its superior features of high sensitivity and non-invasiveness, as well as excellent spatial and temporal fidelity. Herein, we critically summarize the progresses in the intracellular magnesium detection with fluorescent imaging probes. Our discussion focuses on the recent contributions concerning fluorescent imaging probes for mapping magnesium in biological processes. All the candidates are organized according to their acceptor structures. The sensing mechanisms of fluorescent probes are also highly taken into account. Challenges, trends and prospects of fluorescent imaging technology in magnesium detection are also set forth.

Herein, progress in intracellular magnesium detection with fluorescent probes is critically summarized in this work.     

A fluorescence sensing platform of theophylline based on the interaction of RNA aptamer with graphene oxide

RNA, with a structure similar to DNA, should exhibit similar behaviors when it interacts with graphene. In this work, we designed a sensing platform of theophylline based on the interaction of an RNA aptamer with graphene oxide (GO) using the fluorescence as a sensing signal. Firstly, quantum dots (QDs) were modified with the selected ssRNA that can be used as an aptamer to recognize the theophylline. The fluorescence of QDs will be quenched in the presence of GO due to the noncovalent assembly between ssRNA aptamer and GO, leading to fluorescence resonance energy transfer (FRET) from QDs to GO, fluorescence “turn-off”. Then, in the presence of theophylline, the ssRNA aptamer recognizes theophylline to form a dsRNA–theophylline complex. The weak affinity between the complex and GO makes QDs move away from the GO surface, leading to the fluorescence recovery of QDs, fluorescence “turn-on”. Because of the high fluorescence quenching efficiency, unique structure of GO and specificity of the RNA aptamer, the proposed sensing platform exhibits high sensitivity and excellent selectivity for the determination of theophylline. The excellent performance of the sensor based on GO provides new opportunities for sensitive and selective detection of biorecognition events.

A fluorescent sensing platform of theophylline based on the interaction of an RNA aptamer with GO and CdTe as the signal.     

An Au@Ag nanocube based plasmonic nano-sensor for rapid detection of sulfide ions with high sensitivity

Based on the localized surface plasmon resonance (LSPR) technology, a novel plasmonic nanosensor with high sensitivity and high selectivity was prepared for the detection of trace sulfide ions on an individual Au@Ag nanoparticle. Furthermore, it could be used to monitor the sulfurization on an individual Au@Ag nanoparticle surface observed under dark-field microscopy (DFM).

Based on the localized surface plasmon resonance (LSPR) technology, a novel plasmonic nanosensor with high sensitivity and high selectivity was prepared for the detection of trace sulfide ions on an individual Au@Ag nanoparticle.     

Integration of fluorescent probes into metal–organic frameworks for improved performances

Recent years have witnessed a rapid development of fluorescent probes in both analytical sensing and optical imaging. Enormous efforts have been devoted to the regulation of fluorescent probes during their development, such as improving accuracy, sensitivity, selectivity, recyclability and overcoming the aggregation-caused quenching effect. Metal–organic frameworks (MOFs) as a new class of crystalline porous materials possess abundant host–guest chemistry, based on which they display a great application potential in regulating fluorescent probes. This review summarized the research works on the regulation of fluorescent probes using MOFs, with emphasis on the methods of integrating fluorescent probes into MOFs, the regulation effects of MOFs on fluorescent probes, the superiorities of MOFs in regulating fluorescent probes, and the outlook of this subject. It is desirably hoped that this review can provide a useful reference for the researchers interested in this field.

This review surveyed the research works for the regulation of fluorescent probes with metal–organic frameworks based on host–guest chemistry.     

A novel ratiometric AIEE/ESIPT probe for palladium species detection with ultra-sensitivity

Existing fluorescent probes for palladium (Pd) species detection have revealed their vulnerabilities, such as low sensitivity, poor anti-interference ability and long reaction time. In order to develop a faster and more accurate detection method for palladium species at extremely low concentrations, in this study, we designed a novel ratiometric AIEE/ESIPT probe (HPNI-1) based on the Tsuji–Trost reaction for Pd. According to the data obtained, the probe was able to detect Pd species with an ultra-high anti-interference ability (Pd : other metals = 1 : 1000), rapid detection time (within 2 minute) and ratiometric fluorescent signal changes with a 1.34 nM detection limit. This study not only proves that existing methods can be improved but also provides future prospects for HPNI-1 as one of the greatest probes for Pd species detection.

Existing fluorescent probes for palladium (Pd) species detection have revealed their vulnerabilities, such as low sensitivity, poor anti-interference ability and long reaction time.     

N-doped oxidized carbon dots for methanol sensing in alcoholic beverages

Methanol (MeOH) adulteration in alcoholic beverages resulting in irreparable health damage demands highly sensitive and cost-effective sensors for its quantification. As carbon dots are emerging as new biocompatible and sustainable light-emitting detectors, this work demonstrates the hydrothermally prepared nitrogen-doped oxidized carbon dots (NOCDs) as on-off fluorescent nanoprobes to detect MeOH traces in water and alcoholic beverages. The presence of 1% of MeOH in distilled water is found to decrease the NOCD fluorescent emission intensity by more than 90% whereas up to 70% ethanol (EtOH) content changes the signal to within 20% of its initial value. HR-TEM analysis reveals the agglomeration of the nanoprobes suspended in MeOH. Due to their selectivity towards MeOH, the fluorescent nanoprobes were successfully tested using a few MeOH spiked branded and unbranded Mexican alcoholic beverages. Varying degrees of signal quenching is observed from the fluorescent nanoprobes dispersed in different pristine beverages with a detection limit of less than 0.11 v%. Herein, we establish a new perspective towards economically viable non-toxic fluorescent probes as a potential alternative for the detection of MeOH in alcoholic beverages.

Herein, we establish a new perspective towards economically viable non-toxic fluorescent probes as a potential substitute of expensive alternative for the detection of MeOH in alcoholic beverages.     

Adsorption–desorption nano-aptasensors: fluorescent screening assays for ochratoxin A

In this study, a FRET-based fluorescent aptasensor for the detection of ochratoxin A (OTA) was optimized based on the quenching efficiency of single-walled carbon nanotubes (SWCNTs) and the binding affinity of aptamers. OTA aptamers were conjugated with quantum dots and adsorbed to the surface of both acid-modified and unmodified SWCNTs. The maximum fluorescence quenching efficiency of the SWCNTs were compared. Acid-modified SWCNTs (amSWCNTs) have moderate quenching efficiency, providing an optimal sensitivity for qualitative fluorescence-enhancement biosensor assays. The binding parameters of the QD-modified OTA aptamers (1.12.2 and A08min) on the surface of amSWCNTs were compared. Based on our results, the A08min aptamer is a better candidate for OTA detection. Using the A08min aptamer, the SWCNT method had a limit of detection (LOD) of 40 nM. The amSWCNT method had a significantly lower LOD of 14 nM. Turn-on fluorescent nano-aptasensors are emerging as an effective diagnostic tool for simple detection of mycotoxins. Nanocomplexes designed for the detection of mycotoxins in solution and paper-based tests have proven to be useful.

A fluorescent-enhancement biosensor was developed for the mycotoxin ochratoxin A using aptamer-modified quantum dots noncovalently immobilized on carbon nanotubes.     

Orange emissive carbon dots for colorimetric and fluorescent sensing of 2,4,6-trinitrophenol by fluorescence conversion

In this study, infrequent orange carbon nanodots (CNDs) were applied as a dual-readout probe for the effective colorimetric and fluorescent detection of 2,4,6-trinitrophenol (TNP). The orange fluorescence could be rapidly and selectively quenched by TNP, and the colorimetric response from the original pink color to blue could also be captured immediately by the naked eye. A limit of detection of 0.127 μM for TNP was estimated by the fluorescent method and 5 × 10⁻⁵ M by visualized detection. Interestingly, the fluorescence of the CNDs with TNP gradually transitioned from orange to green upon irradiation by a UV lamp, and the colorimetric response transitioned from pink to blue to colorless, which ensured effective multi-response detection of TNP. In addition, the CNDs exhibited bright fluorescence, excellent biocompatibility and low toxicity, making them high-quality fluorescent probes for cellular imaging.

We have described a colorimetric and fluorescent dual-readout probe with a strong and sensitive response towards TNP.     

In situ formation of DNA-templated copper nanoparticles as fluorescent indicator for hydroxylamine detection

Herein, we develop a facile method for selective and sensitive detection of hydroxylamine (HA) based on the in situ formation of DNA templated copper nanoparticles (DNA-CuNPs) as fluorescent probes. It is firstly found that HA as a reducing agent can play a key role in the in situ formation of fluorescent DNA-CuNPs. This special optical property of DNA-CuNPs with (λ_ex = 340 nm, λ_em = 588 nm) with a mega-Stokes shifting (248 nm) makes it applicable for the turn-on detection of HA. In addition, this fluorescent method has several advantages such as being simple, rapid, and environmentally friendly, because it avoids the traditional organic dye molecules and complex procedures. Under optimized conditions, this platform achieves a fluorescent response for HA with a detection limit of 0.022 mM. Especially, successful detection capability in tap waters and ground waters exhibits its potential to be general method.

Herein, we develop a facile method for selective and sensitive detection of hydroxylamine (HA) based on the in situ formation of DNA templated copper nanoparticles (DNA-CuNPs) as fluorescent probes.     

Rational design of fluorescent probe for Hg2+ by changing the chemical bond type

Two kinds of fluorescent probes DFBT and DFABT, and their corresponding water-soluble compounds WDFBT and WDFABT, based on the trimers containing a benzo[2,1,3]thiadiazole moiety and two fluorene moieties are synthesized. Their luminescent behavior towards Hg²⁺ ions and other various metal ions in organic and water solutions are studied in detail via absorption and emission spectroscopy. All these probes show a selective “on–off-type” fluorescent response to Hg²⁺ ions in solution over other metal ions with a maximum detection limit of 10⁻⁷ M. Importantly, the probe type can be changed from irreversible to reversible by altering the bridge mode between the functional units from C C triple bond to C–C single bond. Their detection mechanisms towards Hg²⁺ are studied in detail via mass spectrometry and Job plots, which are attributed to irreversible chemical reaction for DFABT and WDFABT and a reversible coordination reaction for DFBT and WDFBT respectively. Our research results about this kind of organic fluorescent probe provide valuable information to the future design of practical Hg²⁺ fluorescent probes.

Two kinds of fluorescent probes for Hg²⁺ with different detection mechanism have been realized by simply changing the chemical bond.     

Combined in silico and in vitro study of an aptasensor based on citrate-capped AuNPs for naked-eye detection of a critical biomarker of oxidative stress

An elevated level of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) in biosamples has been found to correlate to oxidative stress, and it has been assigned as a critical biomarker of various diseases. Herein, insights into the mechanisms of an aptasensor, based on citrate-capped gold nanoparticles (AuNPs), for 8-oxo-dG detection were elucidated using molecular dynamics (MD) simulations and validated experimentally. We found that the binding mechanism for binding between the anti-8-oxo-dG aptamer and 8-oxo-dG has the following characteristic stages: (i) adsorption stage, (ii) binding stage, and (iii) complex stabilization stage. Our simulations also reveal the binding sites between the anti-8-oxo-dG aptamer and 8-oxo-dG formed through hydrogen bonding during complex formation. A shortened anti-8-oxo-dG-aptamer was also engineered using in silico design, which was expected to improve the analytical performance of the colorimetric aptasensor. The mechanisms of the colorimetric aptasensor in the presence and absence of 8-oxo-dG were also investigated, and found to be in good agreement with the experiments. Complete understanding of the mechanism of the colorimetric aptasensor would open the door for development of novel naked-eye aptasensors.

A visual strategy for 8-oxo-dG monitoring based upon the dispersion of citrate-capped gold nanoparticles has been developed.     

A nanoporous gold-based electrochemical aptasensor for sensitive detection of cocaine

The increasing application of aptamers in bioassays has triggered a lot of research interest for development of highly sensitive and selective sensing platforms. Herein, we report on the design of a sensitive cocaine biosensor by immobilizing the 5′-disulfide-functionalized end of an aptamer sequence on a nanoporous gold (NPG) electrode followed by the conjugation of its 3′-amino-functionalized end to 2,5-dihydroxybenzoic acid (DHBA) as the redox probe. In the presence of cocaine, the aptamer undergoes a conformational change from an open unfolded state to a closed conformation, which reduces the distance between DHBA and the electrode surface, resulting in the enhanced electron-transfer efficiency. Using square wave voltammetric method and under the optimal conditions, the cocaine aptasensor presented two linear responses in the concentration ranges between 0.05–1 and 1–35 μM, with an excellent detection limit of 21 nM. The proposed aptasensor provides a simple and low-cost method for cocaine detection with good reproducibility and accuracy. Furthermore, it could be regarded as a general model to investigate the unique function of aptamer-functionalized nanostructured electrodes to stablish highly advanced electrochemical biosensors for various target analytes of diagnostic importance.

The increasing application of aptamers in bioassays has triggered a lot of research interest for development of highly sensitive and selective sensing platforms.