The hybridisation chain reaction (HCR) is a robust isothermal amplification technique widely used for nucleic acid detection, often paired with silver nanoclusters (AgNCs) for fluorescence-based readouts. However, conventional HCR-AgNC assays are prone to circuit leakage and unpredictable AgNC formation, which can compromise assay reliability. In this study, we present a modular HCR-AgNC hybrid sensor employing a cluster transfer strategy, in which pre-formed AgNCs are introduced separately into the HCR system. This design minimises interference between AgNCs and HCR components, enhancing signal specificity and predictability. Using a DNA analogue of miRNA-141 (DNA-141), a potential prostate cancer biomarker, we demonstrate selective red fluorescence activation upon target recognition. The sensor design was guided by NUPACK simulations, reducing trial-and-error and lowering assay costs. Gel electrophoresis and fluorescence spectroscopy confirmed the system's specificity and sensitivity, achieving a limit of detection of 46 nM. This work establishes a foundational framework for modular and adaptable nucleic acid biosensing, with potential for future sensitivity enhancements and multiplexed detection.

Enzyme-free nucleic acid amplification techniques, such as hybridization chain reaction (HCR), are often coupled with various nanomaterials to fabricate biosensors for nucleic acid amplification and detection, thereby reducing reliance on sophisticated instruments and complicated protocols. A typical HCR system consists of a pair of DNA hairpins (H1 and H2), which unfold and self-assemble into DNA concatemers in the presence of a target nucleic acid. While the versatility of HCR is demonstrated by a variety of available designs, the design process can be tedious, requiring multiple rounds of iteration and optimization to achieve the desired performance. Developing ‘universal’ hairpin designs can significantly streamline this process, reducing the need to redesign HCR hairpin probes for each target. In this work, we developed an enzyme-free and label-free HCR-silver nanoclusters (AgNCs) biosensor using a capture probe (CP-S15) and ‘universal’ hairpins (H1-S13 and H2-S13) for DNA target detection. CP-S15 was designed as a hairpin with a toehold region for DNA target recognition and a loop region to initiate HCR between H1 and H2, which host color-switching fluorescent AgNCs. Notably, H1 and H2 function as ‘universal’ hairpins, as they do not share sequences with or complement the target, making them adaptable for other targets when the CP-S15 loop region remains unchanged. After isothermal HCR incubation with the target and a simple one-pot synthesis of AgNCs at room temperature, the biosensor generated a fluorescence signal, perceived as a color change from red to yellowish orange. The detection range of the biosensor was 50–400 nM, with a detection limit of 41 nM.

In this work, a pair of DNA hairpins was specifically designed for detecting and amplifying a target DNA via hybridization chain reaction (HCR). The biosensor exploits the interactions between gold nanoparticles (AuNPs) and DNA hairpins as well as HCR products, resulting in salt-induced aggregation (SIA) of the AuNPs, observable through visible color changes and UV-vis spectral shifts. This study builds on previous findings regarding the optimal length and sequence of hairpin toeholds and stems, which provide AuNPs with greater protection against SIA. The HCR-AuNP biosensor was developed by applying design guidelines for HCR hairpins and incorporating features that enhance their adsorption onto AuNPs. Intriguingly, hairpins with a 12-14 bp stem and a mixed-base toehold (6-8 nt) containing only a single adenine base provided adequate AuNP stabilization and enabled efficient HCR without leakage. The limit of detection (LOD) for the final HCR-AuNP biosensor was 10.7 nM, with the AuNP color changing from red to blue in the presence of the target DNA. This biosensor is advantageous due to its ease of operation, isothermal nature, and rapid results, all achieved without the need for enzymes or expensive equipment.

Hybridization chain reaction (HCR) and DNA-templated silver nanoclusters (AgNCs) have emerged as powerful tools in biosensing. HCR enables cascade amplification through programmable DNA interactions, while DNA-AgNCs serve as transducing units with unique fluorogenic and electrochemical properties. Integrating these components into a hybrid sensor could significantly enhance sensing capabilities across various fields. Nonetheless, limited studies and the lack of systematic guidelines for HCR-AgNCs systems have hindered research progress, despite their potential. This review aims to address this gap by providing a comprehensive overview of HCR-AgNCs biosensors, facilitating further innovation in this field. The working principles, performance factors, and complementary features are discussed. Thereafter, reported HCR-AgNCs studies are assessed, emphasizing their distinct sensing mechanisms (e.g., fluorogenic, electrochemical), applications across various fields, and challenges in adopting the hybrid sensors. Drawing from the experience developing multiple HCR-AgNCs sensors, insights and guidelines for designing and developing HCR-AgNCs systems are provided for future researchers. Finally, prospective directions in HCR-AgNCs research, including multiplex assays and integration with emerging technologies, are explored to guide future advancements. The synergistic combination of HCR and AgNCs as a hybrid biosensor holds promise for addressing pressing challenges in healthcare, environmental monitoring, and beyond, paving the way for next-generation biosensing technologies.

While fluorescent sensors have been developed for monitoring metal ions in health and diseases, they are limited by the requirement of an excitation light source that can lead to photobleaching and a high autofluorescence background. To address these issues, bioluminescence resonance energy transfer (BRET)-based protein or small molecule sensors have been developed; however, most of them are not highly selective nor generalizable to different metal ions. Taking advantage of the high selectivity and generalizability of DNAzymes, we report herein DNAzyme-based ratiometric sensors for Zn2+ based on BRET. The 8-17 DNAzyme was labeled with luciferase and Cy3. The proximity between luciferase and Cy3 permitted BRET when coelenterazine, the substrate for luciferase, was introduced. Adding samples containing Zn2+ resulted in a cleavage of the substrate strand, causing dehybridization of the DNAzyme construct, thus increasing the distance between Cy3 and luciferase and changing the BRET signals. Using these sensors, we detected Zn2+ in serum samples and achieved Zn2+ detection with a smartphone camera. Moreover, since the BRET pair is not the component that determines the selectivity of the sensors, this sensing platform has the potential to be adapted for the detection of other metal ions with other metal-dependent DNAzymes.

In order to investigate the effects of the secondary coordination sphere in fine-tuning redox potentials (E°') of type 1 blue copper (T1Cu) in cupredoxins, we have introduced M13F, M44F, and G116F mutations both individually and in combination in the secondary coordination sphere of the T1Cu center of azurin (Az) from Pseudomonas aeruginosa. These variants were found to differentially influence the E°' of T1Cu, with M13F Az decreasing E°', M44F Az increasing E°', and G116F Az showing a negligible effect. In addition, combining the M13F and M44F mutations increases E°' by 26 mV relative to WT-Az, which is very close to the combined effect of E°' by each mutation. Furthermore, combining G116F with either M13F or M44F mutation resulted in negative and positive cooperative effects, respectively. Crystal structures of M13F/M44F-Az, M13F/G116F-Az, and M44F/G116F-Az combined with that of G116F-Az reveal these changes arise from steric effects and fine-tuning of hydrogen bond networks around the copper-binding His117 residue. The insights gained from this study would provide another step toward the development of redox-active proteins with tunable redox properties for many biological and biotechnological applications.

This study investigates the effect of DNA hairpins on the stabilization of gold nanoparticles (AuNPs) against salt-induced aggregation (SIA) in label-free colorimetric biosensors. AuNPs were incubated with DNA hairpins of varying stem lengths and toehold sequences, followed by the addition of NaCl, before being subjected to ultraviolet-visible (UV-vis) measurement. Results showed that hairpins with longer stems generally provide better stabilization of AuNPs (18-bp >14-bp >10-bp). No improvement was observed for 14- and 18-bp hairpins with a toehold beyond 8A, which may be attributed to saturated adsorption of hairpins on the gold surface. For 14-bp hairpins with an 8-mer homopolymeric toehold, we observed a stabilization trend of A > C > G > T, similar to the reported trend of ssDNA. For variants containing ≥50% adenine as terminal bases, introducing cytosine or guanine as preceding bases could also result in strong stabilization. As the proportion of adenine decreases, variants with guanine or thymine provide less protection against SIA, especially for guanine-rich hairpins (≥6G) that could form G-quadruplexes. Such findings could serve as guidelines for researchers to design suitable DNA hairpins for label-free AuNP-based biosensors.

A fluorescence biosensor has been developed based on hybridisation chain reaction (HCR) amplification coupled with silver nanoclusters (AgNCs) for nucleic acid detection. The fluorescence was activated via end-to-end transfer of dark AgNCs caged within a DNA template to another DNA sequence that could enhance their red fluorescence emission at 611 nm. Such cluster-transfer approach allows us to introduce fluorogenic AgNCs as external signal transducers, thereby enabling HCR to perform in a predictable manner. The resulted HCR-AgNC biosensor was able to detect target DNA with a detection limit of 3.35 fM, and distinguish the DNA target from single-base mismatch sequences. Moreover, the bright red fluorescence emission was detectable with the naked eye, with concentration of target DNA down to 1 pM. The biosensor also performed well in human serum samples with good recovery. Overall, our cluster-transfer approach provides a good alternative to construct HCR-AgNC assay with less risk of circuit leakage and produce AgNCs in a controllable manner.

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

Gold nanoparticles (AuNPs) have been a popular choice of biosensing material for decades. Given their remarkable physicochemical properties, they have been widely employed in different types of assays, including colorimetric, electrochemical, fluorescence and catalysis. With the emergence of enzyme-free target amplification methods such as hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA), integrating AuNPs with these DNA circuits provide a new paradigm in biosensing. This not only combines the advantages of each component, it also addresses limitations of certain design principles and allows improvement of the assay performance. This review aims to provide a comprehensive reference on different ways to construct AuNPs-based biosensors assisted by HCR and CHA based on different detection principles. For each type of assay, the common design principle is presented and specific features are highlighted that may shed new light on the translation of AuNPs-HCR or AuNPs-CHA biosensors to biologically relevant applications.

Glutathione (GSH) is a useful biomarker in the development, diagnosis and treatment of cancer. However, most of the reported GSH biosensors are expensive, time-consuming and often require complex sample treatment, which limit its biological applications. Herein, a nanobiosensor for the detection of GSH using folic acid-functionalized reduced graphene oxide-modified BSA gold nanoclusters (FA-rGO-BSA/AuNCs) based on the fluorescence quenching interactions is presented. Firstly, a facile and optimized protocol for the fabrication of BSA/AuNCs is developed. Functionalization of rGO with folic acid is performed using EDC/NHS cross-linking reagents, and their interaction after loading with BSA/AuNCs is demonstrated. The formation of FA-rGO, BSA/AuNCs and FA-rGO-BSA/AuNCs are confirmed by the state-of-art characterization techniques. Finally, a fluorescence turn-off sensing strategy is developed using the as-synthesized FA-rGO-BSA/AuNCs for the detection of GSH. The nanobiosensor revealed an excellent sensing performance for the detection of GSH with high sensitivity and desirable selectivity over other potential interfering species. The fluorescence quenching is linearly proportional to the concentration of GSH between 0 and 1.75 µM, with a limit of detection of 0.1 µM under the physiological pH conditions (pH 7.4). Such a sensitive nanobiosensor paves the way to fabricate a “turn-on” or “turn-off” fluorescent sensor for important biomarkers in cancer cells, presenting potential nanotheranostic applications in biological detection and clinical diagnosis.

DNAzymes have emerged as an important class of sensors for a wide variety of metal ions, with florescence DNAzyme sensors as the most widely used in different sensing and imaging applications because of their fast response time, high signal intensity, and high sensitivity. However, the requirements of an external excitation light source and its associated power increase the cost and size of the fluorometer, making it difficult to be used for portable detections. To overcome these limitations, we report herein a DNAzyme sensor that relies on chemiluminescence resonance energy transfer (CRET) without the need for external light. The sensor is constructed by combining the functional motifs from both Pb2+-dependent 8-17 DNAzyme conjugated to fluorescein (FAM) and hemin/G-quadruplex that mimics horseradish peroxidase to catalyze the oxidation of luminol by H2O2 to yield chemiluminescence. In the absence of Pb2+, the hybridization between the enzyme and substrate strands bring the FAM and hemin/G-quadruplex in close proximity, resulting in CRET. The presence of Pb2+ ions can drive the cleavage on the substrate strand, resulting in a sharp decrease in the melting temperature of hybridization and thus separation of the FAM from hemin/G-quadruplex. The liberated CRET pair causes a ratiometric increase in the donor's fluorescent signal and a decrease in the acceptor signal. Using this method, Pb2+ ions have been measured rapidly (<15 min) with a low limit of detection at 5 nM. By removing the requirement of exogenous light excitation, we have demonstrated a simple and portable detection using a smartphone, making the DNAzyme-CRET system suitable for field tests of lake water. Since DNAzymes selective for other metal ions or targets, such as bacteria, can be obtained using in vitro selection, the method reported here opens a new avenue for rapid, portable, and ratiometric detection of many targets in environmental monitoring, food safety, and medical diagnostics.

miRNA (miR)-155 is a potential biomarker for breast cancers. We aimed at developing a nanosensor for miR-155 detection by integrating hybridization chain reaction (HCR) and silver nanoclusters (AgNCs). HCR serves as an enzyme-free and isothermal amplification method, whereas AgNCs provide a built-in fluorogenic detection probe that could simplify the downstream analysis. The two components were integrated by adding a nucleation sequence of AgNCs to the hairpin of HCR. The working principle was based on the influence of microenvironment towards the hosted AgNCs, whereby unfolding of hairpin upon HCR has manipulated the distance between the hosted AgNCs and cytosine-rich toehold region of hairpin. As such, the dominant emission of AgNCs changed from red to yellow in the absence and presence of miR-155, enabling a ratiometric measurement of miR with high sensitivity. The limit of detection (LOD) of our HCR-AgNCs nanosensor is 1.13 fM in buffered solution. We have also tested the assay in diluted serum samples, with comparable LOD of 1.58 fM obtained. This shows the great promise of our HCR-AgNCs nanosensor for clinical application.

Cysteamine-coated gold nanoparticles (cysAuNPs) are positively charged as-synthesised and hence can interact with negatively charged DNA with ease. We have investigated the dependency of the particles' dispersion stage on different concentrations of lysozyme-binding aptamer (LBA). On top of the commonly reported phenomenon where cysAuNPs aggregate as the concentration of LBA increases, we observed that cysAuNPs redispersed after the amount of LBA achieved a certain threshold, dubbed as the critical redispersion concentration (CRC). By harnessing the aggregation and dispersion behaviour of cysAuNPs at LBA below and above the CRC, respectively, we have demonstrated a bimodal colorimetric aptasensor to detect lysozyme as a proof-of-concept study. Apart from being able to quantify the lysozyme in different ranges of concentrations with a visual change in colour, this aptasensor also demonstrated a novel concept of inverse sensitivity (i.e. higher signal with less analyte), leading to a 24-fold higher of signal-to-noise ratio (SNR), in comparison to the conventional sensors. The aptasensor can also selectively distinguish lysozyme and eliminate false results from other control proteins via both modes. The generalisability, as well as potential of cysAuNPs for bimodal colorimetric detection and inverse sensitivity behaviour have made this material an interesting alternative to citrate-coated AuNPs.

A fluorogenic probe has been developed for determination of telomerase activity using chimeric DNA-templated silver nanoclusters (AgNCs). The formation of AgNCs was investigated before (route A) and after (route B) telomerase elongation reaction. Both routes caused selective quenching of the yellow emission of the AgNCs (best measured at excitation/emission wavelength of 470/557 nm) in telomerase-positive samples. The quenching mechanism was studied using synthetically elongated DNA to mimic the telomerase-catalyzed elongation. The findings show that quenching is due to the formation of parallel G-quadruplexes with a –TTA– loop in the telomerase elongated products. The assay was validated using different cancer cell extracts, with intra- and interassay coefficients of variations of <9.8%. The limits of detection for MCF7, RPMI 2650 and HT29 cell lines are 15, 22 and 39 cells/μL. This represents a distinct improvement over the existing telomeric repeat amplification protocol (TRAP) assay in terms of time, sensitivity and cost.

Single strand DNA (ssDNA) chimeras consisting of a silver nanoclusters-nucleating sequence (NC) and an aptamer are widely employed to synthesize functional silver nanoclusters (AgNCs) for sensing purpose. Despite its simplicity, this chimeric-templated AgNCs often leads to undesirable turn-off effect, which may suffer from false positive signals caused by interference. In our effort to elucidate how the relative position of NC and aptamer affects the fluorescence behavior and sensing performance, we systematically formulated these NC and aptamer regions at different position in a DNA chimera. Using adenosine aptamer as a model, we tested the adenosine-induced optical response of each design. We also investigated the effect of linker region connecting NC and aptamer, as well as different NC sequence on the sensing performance. We concluded that locating NC sequence at 5'-end exhibited the best response, with immediate fluorescence enhancement observed over a wide linear range (1-2500 mu M). Our experimental findings help to explain the emission behavior and sensing performance of chimeric conjugates of AgNCs, providing an important means to formulate a better aptasensor. (c) 2018 Elsevier B.V. All rights reserved.

AP-2 gamma (AP-2γ) is a transcription factor that plays pivotal roles in breast cancer biology. To search for small molecule inhibitors of AP-2γ, we performed a high-throughput fluorescence anisotropy screen and identified a polyoxometalate compound with Wells–Dawson structure K6[P2Mo18O62] (Dawson-POM) that blocks the DNA-binding activity of AP-2γ. We showed that this blocking activity is due to the direct binding of Dawson-POM to AP-2γ. We also provided evidence to show that Dawson-POM decreases AP-2γ-dependent transcription similar to silencing the gene. Finally, we demonstrated that Dawson-POM contains anti-proliferative and pro-apoptotic effects in breast cancer cells. In summary, we identified the first small molecule inhibitor of AP-2γ and showed Dawson-POM-mediated inhibition of AP-2γ as a potential avenue for cancer therapy.

Significance Nature spent millions of years to evolve electron transfer proteins that span a wide range of reduction potentials (E°′) under physiological conditions, from −1 V to 1 V vs. standard hydrogen electrode. Understanding the rules that govern such tuning within similar classes of metalloproteins enables scientists to rationally tune the E°′of their catalysts without changing the active site. An ultimate test of such understanding is to achieve the entire range of E°′ within a single protein, a feat that has not been achieved yet. Herein, we conclusively found that we can cover the entire 2-V range of E°′ using a single protein with five mutations and two metal ions. We have also provided explanations for structural features responsible for such high potential.

12 years after the introduction of DNA-templated silver nanoclusters (DNA-AgNCs), exciting progress has been made and yet we are still in the midst of trying to fully understand this nanomaterial. The prominent excellence of DNA-AgNCs is undoubtedly its modulatable emission property, of which how variation in DNA templates causes emission tuning remains elusive. Based on the up-to-date DNA-AgNCs, we aim to establish the correlation between the structure/sequence of DNA templates and emission behaviour of AgNCs. Herein, we systematically present a wide-range of DNA-AgNCs based on the structural complexity of the DNA templates, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), triple-stranded DNA (tsDNA) and DNA nanostructures. For each DNA category, we discuss the emission property, quantum yield and synthesis condition of the respective AgNCs, before cross-comparing the impact of different DNA scaffolds on the properties of AgNCs. A future outlook for this area is given as a conclusion. By putting the information together, this review may shed new light on understanding DNA-AgNCs while we are expecting continuous breakthroughs in this field.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 °C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.