The optimal sequence for synthesizing copper nanoclusters is a promising research area. Initially, random dsDNA sequences yielded low fluorescence intensity, which constrained visual detection under UV light. Poly-AT dsDNA sequences later produced visible fluorescence, but it caused significant interference in negative samples when combined with gene amplification techniques. This interference occurs because the single-stranded poly-AT primer can self-anneal into a double-stranded AT sequence, efficiently synthesizing copper nanoclusters. To mitigate this, we designed a poly-AAT sequence at the primer's 5' end, creating a single base pair mismatch every three nucleotides during self-annealing. This adjustment reduced synthesis efficiency of copper nanoclusters in negative samples, improving the visual distinction between negative and positive results. We applied this method to identify the HLA-B*5801 gene, thereby demonstrating its efficacy even within a GC-rich region of human genomic DNA. Our method showed 100% agreement with a commercial qPCR kit, with results distinguishable under UV light. We conclude that the poly-AAT sequence is more suitable for integrating copper nanoclusters synthesis with nucleic acid amplification detection techniques, with potential applications in microelectronics, biosensing, and catalysis.

The development of simple and versatile approaches for the fabrication of DNA-based composite nanomaterials, endowed with defined morphologies and specific functionalities, is of paramount importance for various applications. Herein, we report a simple approach for the synthesis of multifunctional copper-DNA nanoflowers (Cu-DNF) that exclusively consist of rolling circle polymerized nanoflowers (DNF) and in situ synthesized concatemeric fluorescence copper nanoparticles. Through meticulous regulation of the assembly process, it is possible to generate Cu-DNF with precise sizes and stable fluorescence properties. The obtained Cu-DNF possesses robust biostability to resist degradation by nuclease, presumably resulting from the dense structure of the Cu-DNF. The Cu-DNF were also encoded with polyvalent tandem CD63 aptamer sequences, which enhanced their binding affinity and internalization efficiency into tumor cells. We demonstrate that the multifunctional Cu-DNF can efficiently internalize tumor cells for tracking and imaging analysis of intracellular microRNA. This approach may be beneficial for creating multifunctional DNA-based composite nanomaterials for various technological applications.

Self-assembly of nanoparticles (NPs) in solution has garnered tremendous attention among researchers because of their electrical, chemical, and optoelectronic properties at the macroscale with potential applications in bio-imaging, bio-medicine, and therapeutics. Control of size, shape, and composition at the nanoscale is important in tuning the material's bulk properties. The grafting of NPs with polymers enables us to tune such bulk material properties at the nano level by controlling their assemblies, especially in solutions. The stiffness of grafts plays a crucial role in tuning the self-assembly of spherical NPs grafted with polyions (PGNs). Many recent studies based on single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) showed the potential applications of such assemblies. In this work, we have performed coarse-grained molecular dynamics (MD) simulations to understand the charge-driven self-assembly of PGNs by varying stiffness of polymer grafts, the grafting density, and graft length. Self-assembly of these PGNs leads to the formation of different structures driven by the rigidity of polyion chains and the electrostatic interactions. A dramatic change in morphological transitions can be achieved, ranging from rings, strings, and percolated structures and ordered to disordered aggregates by tuning the control parameters. The percolated structures form disordered structures upon annealing with potential applications in thermal under filling, neuromorphic devices, and biological systems including drug delivery, and therapeutics.

After the discovery of DNA during the mid-20th century, a multitude of novel methodologies have surfaced which exploit DNA for its various properties. One such recently developed application of DNA is as a template in metal nanocluster formation. In the early years of the new millennium, a group of researchers found that DNA can be adopted as a template for the binding of metal nanoparticles that ultimately form nanoclusters. Three metal nanoclusters have been studied so far, including silver, gold, and copper, which have a plethora of biological applications. This review focuses on the synthesis, mechanisms, and novel applications of DNA-templated metal nanoclusters, including the therapies that have employed them for their wide range of fluorescent properties, and the future perspectives related to their development by exploiting machine learning algorithms and molecular dynamics simulation studies.

To address the food safety issues caused by toxins, we established a fluorescent copper nanocluster biosensor based on magnetic aptamer for the visual and quantitative detection of ZEN. Specifically, we utilized the docking-aided rational tailoring (DART) strategy to analyze intermolecular force and interaction sites between zearalenone (ZEN) and the aptamer, and optimize the long-chain aptamer step by step to enhance the binding affinity by 3.4 times. The magnetic bead-modified aptamer underwent conformational changes when competing with complementary sequences to bind with ZEN. Then, the released complementary sequences will be amplified in template-free mode with the presence of the terminal deoxynucleotidyl transferase (TdT), and generating T-rich sequences as the core sequences for the luminescence of copper nanoclusters. The luminescence could be visualized and quantitatively detected through ultraviolet irradiation. The proposed label-free aptasensor exhibited high sensitivity and specificity, with a low limit of detection (LOD) of 0.1 ng/mL.

This study presented a nanoparticle-enhanced aptamer-recognizing homogeneous detection system combined with a portable instrument (NASPI) to quantify lipoarabinomannan (LAM). This system leveraged the high binding affinity of aptamers, the high sensitivity of nanoparticle cascade amplification, and the stabilization effect of dual stabilizers (fructose and histone), and used probe-Cu2+ to achieve LAM detection at concentrations ranging from 10 ag mL-1 to 100 fg mL-1, with a limit of detection of 3 ag mL-1 using a fluorometer. It can also be detected using an independently developed handheld fluorometer or the red-green-blue (RGB) camera of a smartphone, with a minimum detection concentration of 10 ag mL-1. We validated the clinical utility of the biosensor by testing the LAM in the urine of patients. Forty urine samples were tested, with positive LAM results in the urine of 18/20 tuberculosis (TB) cases and negative results in the urine of 6/10 latent tuberculosis infection cases and 10/10 non-TB cases. The assay results revealed a 100% specificity and a 90% sensitivity, with an area under the curve of 0.9. We believe that the NASPI biosensor can be a promising clinical tool with great potential to convert LAM into clinical indicators for TB patients.

CRISPR-Cas12a with robust trans-cleavage activity were employed to mitigate background fluorescence signal, achieving sensitive detection of miRNA-21. The activation of trans-cleavage activity of Cas12a was achieved by utilizing cDNA as a trigger. Upon the presence of target miRNA-21, cDNA hybridizes with it forming a DNA/RNA double-stranded structure. Exonuclease III (ExoIII) facilitates the degradation of cDNA, releasing the target for subsequent cycles. Due to cDNA degradation, the trans-cleavage activity of Cas12a remains unactivated and does not disrupt the synthesis template of copper nanoparticles. Addition of Cu2+ and AA leads to the formation of highly fluorescent copper nanoparticles. Conversely, in absence of miRNA-21, intact cDNA activates trans-cleavage activity of Cas12a, resulting in degradation of the synthesis template and failure in synthesizing fluorescent copper nanoparticles. This method exhibits excellent selectivity with a low limit of detection (LOD) at 5 pM. Furthermore, we successfully applied this approach to determine miRNA-21 in cell lysates and human serum samples, providing a new approach for sensitive determination of biomarkers in biochemical research and disease diagnosis.

Hybrid materials that combine organic polymers and biomacromolecules offer unique opportunities for precisely controlling 3D chemical environments. Although biological or organic templates have been separately used to control the growth of inorganic nanoclusters, hybrid structures represent a relatively unexplored approach to tailoring nanocluster properties. Here, we demonstrate that a molecularly defined lysozyme-polymer resin material acts as a structural scaffold for the synthesis of copper nanoclusters (CuNCs) with well controlled size distributions. The resulting CuNCs have significantly enhanced fluorescence compared with syntheses based on polymeric or biological templates alone. The synergistic approach described here is appealing for the synthesis of biocompatible fluorescent labels with improved photostability.

Bioassays using inductively coupled plasma mass spectrometry (ICP-MS) have gained increasing attention because of the high sensitivity of ICP-MS and the various strategies of labeling biomolecules with detectable metal tags. The classic strategy to tag the target biomolecules is through direct antibody-antigen interaction and DNA hybridization, and requires the separation of the bound from the unbound tags. Label-free ICP-MS techniques for biomolecular assays do not require direct labeling: they generate detectable metal ions indirectly from specific biomolecular reactions, such as enzymatic cleavage. Here, we highlight the development of three main strategies of label-free ICP-MS assays for biomolecules: (1) enzymatic cleavage of metal-labeled substrates, (2) release of immobilized metal ions from the DNA backbone, and (3) nucleic acid amplification-assisted aggregation and release of metal tags to achieve amplified detection. We briefly describe the fundamental basis of these label-free ICP-MS assays and discuss the benefits and drawbacks of various designs. Future research is needed to reduce non-specific adsorption and minimize background and interference. Analytical innovations are also required to confront challenges faced by in vivo applications.

In this study, we systematically investigated the interactions between Cu2+ and various biomolecules, including double-stranded DNA, Y-shaped DNA nanospheres, the double strand of the hybridization chain reaction (HCR), the network structure of cross-linked HCR (cHCR), and small molecules (PPi and His), using Cu2+ as an illustrative example. Our research demonstrated that the coordination between Cu2+ and these biomolecules not only is suitable for modulating luminescent material signals through complexation reactions with Cu2+ but also enhances signal intensities in materials based on chemical reactions by increasing spatial site resistance and local concentration. Building upon these findings, we harnessed the potential for signal amplification in self-assembled DNA nanospheres and the selective complexation modulation of calcein in conjunction with the aptamer targeting mucin 1 as a recognition probe. We applied this approach to the analysis of circulating tumor cells, with the lung cancer cell line A549 serving as a representative model. Our assay, utilizing both a fluorometer and a handheld detector, achieved impressive detection limits of ag/ml and single-cell levels for mucin 1 and A549 cells, and this approach was successfully validated using 46 clinical samples, yielding 100% specificity and 86.5% sensitivity. Consequently, our strategy has paved the way for more portable and precise disease diagnosis.

The excessive secretion of H2O2 within cells is closely associated with cellular dysfunction. Therefore, high sensitivity in situ detection of H2O2 released from living cells was valuable in clinical diagnosis. In the present work, a novel electrochemical cells sensing platform by synthesizing copper nanoclusters (CuNCs) at room temperature based on DNA nanoribbon (DNR) as a template (DNR-CuNCs). The tight and ordered arrangement of nanostructured assemblies of DNR-CuNCs conferred the sensor with superior stability (45 days) and electrochemical performance. The MUC1 aptamer extending from the DNR template enabled the direct capture MCF-7 cells on electrode surface, this facilitated real-time monitoring of H2O2 release from stimulated MCF-7 cells. While the captured MCF-7 cells on the electrode surface significantly amplified the current signal of H2O2 release compared with the traditional electrochemical detection H2O2 released signal by MCF-7 cells in PBS solution. The approach provides an effective strategy for the design of versatile sensors and achieving monitored cell release of H2O2 in long time horizon (10 h). Thereby expanding the possibilities for detecting biomolecules from live cells in clinical diagnosis and biomedical applications.

Food safety concerns have become a significant threat to human health and well-being, catching global attention in recent years. As a result, it is imperative to research conceptually novel biosensing and effective techniques for food matrices detection. Currently, DNA-templated metal nanoclusters (DNA-MNCs) are considered as one of the most promising nanomaterials due to their excellent properties in biosensing. While DNA-MNCs have garnered increasing interest, the reviews of design strategies, applications, and futuristic prospects for biosensing have been hardly found especially in food safety. The synthesis of DNA-MNCs and their use as biosensing materials in food contamination detection, including pathogenic bacteria, toxins, heavy metals, residues of pesticides, and others were comprehensively reviewed. In addition, we summarize the properties of DNA-MNCs briefly and discuss the challenges and future trends. The application of DNA-MNCs powered biosensing has been demonstrated and actively studied, which is a promising paradigm for food safety testing that can supplement or even replace current existing methods. Despite the challenges of difficulty regulating accurately, poor stability, low quantum yield, and difficult commercial transformation, the application prospects of DNA-MNCs biosensors are promising. This review aims to provide insights and directions for the future development of DNA-MNCs based food detection technology.

Nanoclusters (NCs) are meticulously small, kinetically stable, crystalline materials which hold immense potential as multifaceted catalysts for a broad range of biomedical applications. Metal NCs are atomically precise and exist within the range of Fermi wavelength of electrons. They are highly advantageous as functional materials as their physicochemical properties can be customized to meet specific requirements. Copper NCs (CuNCs) are emerging as an efficient substitute to the other existing metal NCs. The synthesis of CuNCs is highly methodical, fast, cost effective and does not involve any complicated manipulation. On the contrary to gold and silver NCs, copper is a vital trace element for humans that can be excreted easily out the body. Further, the relatively inexpensiveness and easy availability of copper aids in potential nanotechnological applications in large quantity. As such, CuNCs have attracted great interest among the research community recently. The modern developments in the strategy, synthesis, surface modifications, and use of CuNCs in diagnosis of disease, imaging and treatment have been discussed in the present review. Approaches to regulate and augment the emission of CuNCs, challenges and drawbacks have also been considered. This review brings to light the multifarious applications of CuNCs and their potential as emerging theranostic agents. It is anticipated that the visions and directions for translating existing developments in CuNCs from the laboratory to the clinic can be further improved and enhanced.

Ligand-RNA interaction assay provides the basis for developing new RNA-binding small molecules. In this study, fluorescent copper nanoclusters (CuNCs) were first prepared using two kinds of HIV-1 RNA targets, rev-responsive element (RRE) and transactivator response element (TAR) RNA, as new templates, and it was found that the fluorescence of the single RNA-templated CuNCs was negligible. Using neomycin as a model drug, the fluorescence could be augmented (approximately 6 times) for the neomycin/RNA-templated CuNCs. Thus, a novel method was developed for ligand-RNA interactions by observing the fluorescence changes in CuNCs prepared using RNA before and after the addition of ligands. The preparation parameters of neomycin/RNA-CuNCs were optimized. The as-prepared CuNCs were characterized using UV-vis spectroscopy, fluorescence spectroscopy, and high-resolution transmission electron microscope. Circular dichroism spectral analysis showed that RRE and TAR were inclined to form a double-stranded structure after interaction with neomycin, which was more conducive to the formation of CuNCs. The interactions of neomycin and three test drugs (amikacin, gentamicin, and tobramycin) with RNA were investigated using the proposed method, and the binding constants and number of binding sites were obtained through theoretical calculations. This study provides a novel approach for ligand-RNA interaction assays.

A multimodal analytical strategy utilizing different modalities to cross-validate each other, can effectively minimize false positives or negatives and ensure the accuracy of detection results. Herein, we establish a colorimetric, photothermal, and fluorescent triple modal CRISPR/Cas12a detection platform (CPF-CRISPR). An MNPs-ssDNA-HRP signal probe is designed to act as a substrate to trigger three signal outputs. In the presence of the DNA target, MNPs-ssDNA-HRP is cleaved by the activated CRISPR/Cas12a, resulting in the release of HRP and generating short DNA strands with 3-terminal hydroxyl on magnetic beads. The released HRP subsequently catalyzed TMB-H2O2 reaction and oxidized TMB is used for colorimetric and photothermal signal detection. Under the catalysis of terminal deoxynucleotidyl transferase (TdT), the remaining short DNA strands are used as primers to form poly-T and function as scaffolds to form copper nanoclusters for fluorescent signal output. To verify the practical application of CPF-CRISPR, we employed MRSA as a model. The results demonstrate the platform's high accuracy and sensitivity, with a limit of detection of 101 CFU/mL when combined with recombinase polymerase amplification. Therefore, by harnessing the programmability of CRISPR/Cas12a, the biosensor has the potential to detect various drug-resistant bacteria, demonstrating significant practical applicability.

A sensitive fluorescent sensor has been developed for the determination of tetracycline (TC) using adenine thymine (AT)-rich single-stranded DNA (ssDNA) templated copper nanoclusters (CuNCs) as a fluorescent probe. Fluorescent ssDNA-CuNCs were synthesized by employing AT-rich ssDNA as templates and ascorbic acid as reducing agents through a facile one-step method. The as-prepared ssDNA-CuNCs exhibited strong fluorescence with a large Stokes shift (240 nm) and stable fluorescence emission. In the presence of TC, the fluorescent intensity of ssDNA-CuNCs was obviously decreased through the inner filter effect, due to the spectral overlapping between ssDNA-CuNCs and TC. Under the optimal conditions, the strategy exhibited sensitive detection of TC with a linear range from 2 nM to 30 μM and with a limit of detection of 0.5 nM. Furthermore, the sensor was successfully applied for the detection of TC in milk samples. Therefore, it provided a simple, rapid, and label-free fluorescent method for TC detection.

Escherichia coli O157:H7 poses a threat to humans. Traditional ELISA is not a sensitive method for the detection of E. coli O157:H7. Here, an efficient method was designed for improving the load capacity of alkaline phosphatase (ALP) with streptavidin scaffolded DNA tetrad (SS-DNAt). With more ALP, more ascorbic acid 2-phosphate was catalyzed to ascorbic acid that was used to synthesize fluorescence poly adenine-thymine-templated copper nanoclusters. Based on SS-DNAt, fluorescence ELISA was successfully proposed for improving the sensitivity for detection of E. coli O157:H7 in milk samples. The method showed a linear range of 104 to 106 cfu/mL. The limit of detection of fluorescence ELISA was 3.75 × 103 cfu/mL and 6.16-fold better than that of traditional ELISA. The recovery of the fluorescence ELISA was 86.7 to 93.6% with the coefficient of variation of 5.6 to 10.5% in milk. This method could be used to detect hazardous material in food.

At present, a large number of studies have demonstrated that miRNAs can be used as biological indicators for the diagnosis and treatment of diseases such as tumours and cancer, so it is important to develop a new miRNA detection platform. In this work, miRNA-122 is used as the basis for targeting detection agents. We have designed an unlabelled DNA1 that undergoes partial hybridisation and has a 20 T base long strand. The fluorescent signal in this experiment is derived from copper nanoclusters (CuNCs) generated on the circular T-long strand of DNA1. At the same time, DNA1 is able to react with miRNA-122 and achieve hydrolysis of the part bound to miRNA-122 via the action of nucleic acid exonuclease III (Exo III), leaving a part of the DNA, called DNA3, while releasing miRNA-122 to participate in the next reaction, thus achieving circular amplification. DNA3 is able to react with DNA2, which is bound to streptavidin magnetic beads (SIBs) and separated from the reaction solution via the application of a magnetic field. Overall, this is a fluorescence signal reduction experiment, and the strength of the fluorescence signal from the copper nanoclusters can determine whether the target miRNA-122 is present or not. The degree of fluorescence reduction indicates how much DNA1, and thus the amount of target miRNA-122, has been hydrolysed. By evaluating the variations in the fluorescence signal under optimised conditions, we discovered that this method has good sensitivity, with a detection limit as low as 0.46 nM, better than many other previous works on fluorescence signal-based biosensors for miRNA detection. This technique offers high discrimination and selectivity and can serve as a persuasive reference for early diagnosis.

Escherichia coli is the major pathogen that causes bloodstream infections (BSI). It is critical to develop nonculture identification methods which can meet the urgent need of clinical diagnosis and treatment. In this study, we reported a homogeneous fluorescence E. coli analysis system using β-galactosidase (β-Gal) as the biomarker and double-stranded DNA-templated copper nanoparticles (dsDNA-Cu NPs) as the signal output. The product of the enzymatic hydrolysis reaction, p-aminophenol (PAP), could reduce Cu²⁺ to Cu⁺, triggering the alkyne-azido cycloaddition reaction (CuAAC). Subsequently, the hybrid chain reaction (HCR) was initiated, producing the dsDNA template used to generate Cu NPs in situ. The system achieved a wide linear range for β-Gal and E. coli 1-10⁴ mU/L and 10^-2-10 colony-forming unit (CFU)/mL, and a detection limit of 0.3 mU/L and 0.003 CFU/mL, respectively. 65 samples (45 blood and 20 urine) were collected to evaluate the clinical practicality. The results demonstrated remarkable area under the curve (AUC) values of 0.95 and 0.916 from uncultured urine and blood, respectively. It had 100% specificity and 83.3% sensitivity. The whole duration of the strategy was 3.5 h, which significantly reduced the turnaround time (TAT) and facilitated early BSI diagnosis to improve patients' prognosis. Our work had the potential to be an alternative to culture-based methods in clinics.

Lipoarabinomannan (LAM) is a prospective noninvasive biomarker for tuberculosis (TB) diagnosis. Here, we report a visual immunoassay of high sensitivity for detecting LAM in urine samples toward TB diagnosis. This method uses a DNA-linked immunosorbent of LAM, followed by a transduction cascade into amplified visual signals using quantum dots (QDs) and calcein reaction with Cu²⁺ and copper nanoparticles (Cu NPs). The limit of detection (LOD) for LAM in the urine reaches 2.5 fg/mL and 25 fg/mL using a fluorometer and length readouts on strips, respectively, demonstrating an ultrahigh sensitivity. The clinical validation of the proposed assay was performed with 147 HIV-negative clinical urine specimens. The results show the sensitivity of test is 94.1% (16/17) for confirmed TB (culture-positive) and 85% (51/60) for unconfirmed TB (clinical diagnosis without positive culture results), respectively, when the test cutoff value is 40 fg/mL for TB. Its specificity is 89.2% (25/28) in non-TB and nontuberculous mycobacterial patients. The area under the curve (AUC) was 0.86 when controls were non-TB and LTBI patients, while the AUC was 0.92 when controls were only non-TB patients. This highly sensitive visual immunoassay of LAM has shown potential for noninvasive diagnosis of TB using urine samples.

A fluorescent method is described for trypsin determination through the strong electrostatic interactions between cationic polyelectrolytes and single-stranded DNA (ssDNA) templated Au nanoclusters (AuNCs). The ssDNA-AuNCs display improved fluorescence emission with excitation/emission maxima at 280/475 nm after being incorporated with poly(diallyldimethylammonium chloride) (PDDA). Fluorescent enhancement is mainly attributed to the electrostatic interactions occurring  between PDDA and ssDNA templates. This can make the conformation of the ssDNA templates to change. Thus, it offers a better microenvironment for stabilizing and protecting ssDNA-AuNCs, and results in fluorescence emission enhancement. By using protamine as a model, the method is employed for the determination of trypsin. The assay enables trypsin to be determined with good sensitivity and a linear response ranging from 5 ng⋅mL^-1 to 60 ng⋅mL^-1 with a 1.5 ng⋅mL^-1 limit of detection. It is also extended to determine  the trypsin contents in human's serum samples with recoveries between 98.7% and 103.5% with relative standard deviations (RSDs) between 3.5% and 4.8%. A novel fluorescent strategy has been developed for of trypsin determination by using protamine mediated fluorescent enhancement of DNA templated Au nanoclusters.

Two-dimensional (2D) amorphous materials could outperform their crystalline counterparts toward various applications because they have more defects and reactive sites and thus could exhibit a unique surface chemical state and provide an advanced electron/ion transport path. Nevertheless, it is challenging to fabricate ultrathin and large-sized 2D amorphous metallic nanomaterials in a mild and controllable manner due to the strong metallic bonds between metal atoms. Here, we reported a simple yet fast (10 min) DNA nanosheet (DNS)-templated method to synthesize micron-scale amorphous copper nanosheets (CuNSs) with a thickness of 1.9 ± 0.4 nm in aqueous solution at room temperature. We demonstrated the amorphous feature of the DNS/CuNSs by transmission electron microscopy (TEM) and X-ray diffraction (XRD). Interestingly, we found that they could transform to crystalline forms under continuous electron beam irradiation. Of note, the amorphous DNS/CuNSs exhibited much stronger photoemission (∼62-fold) and photostability than dsDNA-templated discrete Cu nanoclusters due to the elevation of both the conduction band (CB) and valence band (VB). Such ultrathin amorphous DNS/CuNSs hold great potential for practical applications in biosensing, nanodevices, and photodevices.

Many current chemodynamic therapy (CDT) strategies suffer from either low therapeutic efficiency or the deficiency of poor targeting. The low therapeutic efficiency is mainly ascribed to the intracellular antioxidant system and the inefficient Fenton reaction in the weakly acidic tumor microenvironment (TME). Herein, by exploitation of the diverse function and programmability of functional nucleic acid, aptamer-tethered nanotrains of DNA copper nanoclusters (aptNTDNA-CuNCs) were assembled to simultaneously achieve targeted recognition, loading, and delivery of CDT reagents into tumor cells without an external carrier. The intracellular hydrogen peroxide (H₂O₂) oxidized nanotrains of DNA-CuNCs to produce a lot of Cu²⁺ and Cu⁺ ions, which can generate reactive oxygen species (ROS) in the weakly acidic TME based on the pH-independent Fenton-like reaction of Cu⁺/H₂O₂. Meanwhile, the redox reaction between intracellular glutathione (GSH) and Cu²⁺ depleted GSH and generated Cu⁺ ions, which weakened the antioxidant ability of cancer cells and further enhanced the Fenton-like reaction of Cu⁺/H₂O₂, respectively. Thus, the cascade Fenton-like reaction and GSH depletion doubly improved the efficacy of CDT. The in vivo and in vitro study solidly confirmed that aptNTDNA-CuNCs have excellent antitumor efficacy and no cytotoxicity to healthy cells. Therefore, aptNTDNA-CuNCs can act as CDT reagents to achieve highly efficient, biocompatible, and targeted CDT.

Here, based on the design of rational primers and copper nanoclusters (CuNCs), we present a method for the accurate detection of the SARS-CoV-2 Delta variant, which is capable of distinguishing the Delta variant with its single nucleotide polymorphism from the 'wild type' coronavirus (NC_045512.2), and realizing visualization signal out. Specifically, we show that dual priming oligonucleotide (DPO) primers and AT primers can be used to distinguish between wild types and mutations of this virus by polymerase chain reaction (PCR) analysis and that visualization can be achieved via the red fluorescence of CuNCs in ultraviolet radiation. Among the results, it was found that the R-1-down (DPO)-6I and F-1-30 AT, with the single nucleotide deletion site designed at the 3' end of the downstream primer, showed the best specificity towards the Delta variant. Moreover, the use of AT primers increased the AT contents of the PCR products, thus meeting the template requirements generated by the CuNCs. It was also found that the AT primers could assist with improving detection specificity. Finally, we demonstrate that the visualization of the CuNCs-based detection assay exhibited a linear relationship in 0.5 pg μL^-1-50 ng μL^-1, with a limit of quantitation (LOQ) of 0.5 pg μL^-1.

A rapid and convenient fluorescence glyphosate (GLYP) biosensor was developed based on DNA-templated copper nanoparticles (DNA-CuNPs). In the absence of GLYP, the DNA-CuNPs were formed through the reduction of Cu2+ by vitamin C (Vc). The DNA-CuNPs emitted intense fluorescence at 615 nm when being excited at 340 nm. In the presence of GLYP, GLYP can strongly chelate with Cu2+ by the phosphate and carboxyl groups to decrease the amount of free Cu2+. Due to the lack of free Cu2+, DNA-CuNPs cannot be formed, which caused the fluorescence to decrease. The whole detection process of this proposed GLYP biosensor can be completed within 14 min. Titration experiments showed that this biosensor had a linear relationship for GLYP in the range 1 to 18 µM with a limit of detection (LOD) of 0.47 µM. This biosensor showed obvious selectivity among other pesticides, even between GLYP and organophosphorus pesticides. This biosensor performed well for GLYP detection in real samples with recoveries of 88.0-104.0%.

A new preparation route for high-luminescent blue-emission pepsin copper nanoclusters (Pep-CuNCs) is introduced in this work. The synthesized nanoclusters are based on a pepsin molecule, which is a stomach enzyme that works to digest proteins that exist in undigested food. Here, we have developed an eco-friendly technique through microwave-assisted fast synthesis. The resulting copper nanoclusters (CuNCs) exhibit significant selectivity towards Pb(II) ions. The pepsin molecule was utilized as a stabilizer and reducing agent in the production procedure of Pep-CuNCs. The characteristics of the resulting Pep-CuNCs were studied in terms of size, surface modification, and composition using various sophisticated techniques. The CuNCs responded to Pb(II) ions through the fluorescence quenching mechanism of the CuNCs' fluorescence. Thus, great selectivity of Pep-CuNCs towards Pb(II) ions was observed, allowing sensitive determination of this metal ion at lab-scale and in the environment. The CuNCs have detection limits for Pb(II) in very tenuous concentration at a nanomalar scale (11.54 nM). The resulting Pep-CuNCs were utilized significantly to detect Pb(II) ions in environmental samples. Additionally, the activity of Pep-CuNCs on different human tumor cell lines was investigated. The data for the observed behavior indicate that the Pep-CuNCs displayed their activity against cancer cells in a dose dependent manner against most utilized cancer cell lines.

Mercuric (Hg²⁺) ions released from human activities, natural phenomena, and industrial sources are regarded as the global pollutant of world's water. Hg²⁺ ions contaminated water has several adverse effects on human health and the environment even at low concentrations. Therefore, rapid and cost-effective method is urgently required for the detection of Hg²⁺ ions in water. Although, the current analytical methods applied for the detection of Hg²⁺ ions provide low detection limit, they are time consuming, require expensive equipment, and are not suitable for in-situ analysis. Metal nanoclusters (MNCs) consisting of several to ten metal atoms are important transition missing between single atoms and plasmonic metal nanoparticles. In addition, sub-nanometer sized MNCs possess unique electronic structures and the subsequent unusual optical, physical, and chemical properties. Because of these novel properties, MNCs as a promising material have attracted considerable attention for the construction of selective and sensitive sensors to monitor water quality. Hence this review is focused on recent advances on synthesis strategies, and optical and chemical properties of various MNCs including their applications to develop optical assay for Hg²⁺ ions in aqueous solutions.

Here, we report a simple aptamer-based toxoid test with both fluorescence and binary visual readouts. This test is established based on our recent finding that CdTe quantum dots could differentiate DNA templated Cu NPs from Cu²⁺. Through the further integration with enzyme-free triple parallel hybridization chain reaction, cation exchange reaction, and inkjet printing, we demonstrated specific detection of tetanus toxoid with a limit-of-detection (LOD) of 0.25 fg/mL using fluorescence readout. Using color- and distance-based binary visual readouts, we were able to achieve LODs of 10 fg/mL and 1 fg/mL, respectively. The quantitative test results for tetanus toxoid using both fluorescence and visual readouts were successfully validated in 84 clinical serum samples. Moreover, our strategy also enabled accurate monitoring of tetanus toxoid levels in patients before and after drug treatment. On the basis of our clinical test results, we recommend a cutoff value of 5 fg/mL for tetanus infection.

Because of high sensitivity and specificity, isothermal nucleic acid amplification are widely applied in many fields. To facilitate and improve their performance, various nanomaterials, like nanoparticles, nanowires, nanosheets, nanotubes, and nanoporous films are introduced in isothermal nucleic acid amplification. However, the specific application, roles, and prospect of nanomaterials in isothermal nucleic acid amplification have not been comprehensively reviewed. Here, the application of different nanomaterials (0D, 1D, 2D, and 3D) in isothermal nucleic acid amplification is comprehensively discussed and recent progress in the field is summarized. The nanomaterials are mainly used for reaction enhancer, signal generation/amplification, or surface loading carriers. In addition, 3D nanomaterials can be also functioned as isolated chambers for digital nucleic acid amplification and the tools for DNA sequencing of amplified products. Challenges and future recommendations are also proposed to be better used for recent covid-19 detection, point-of-care diagnostic, food safety, and other fields.

The development of powerful, simple and cost-effective signal amplifiers has significant implications for biological research and analysis. Hybridization chain reaction (HCR) has attracted increasing attention because of its enzyme-free, simple, and efficient amplification. In the HCR process, an initiator probe triggered a pair of metastable hairpins through a cross-opening process to propagate a chain reaction of hybridization events, yielding a long-nicked double-stranded nucleic acid structure. To achieve more noticeable signal amplification, nanomaterials, including graphene oxide, quantum dots, gold, silver, magnetic, and other nanoparticles, were integrated with HCR. Various types of colorimetric, fluorescence, plasmonic analyses or chemiluminescence optical sensing strategies incorporating nanomaterials have been developed to analyze various targets, such as nucleic acids, small biomolecules, proteins, and metal ions. This review summarized the recent advances of HCR technology pairing diverse nanomaterials in optical detection and discussed their challenges.

The development and application of metal nanoclusters (MNCs) in nucleic acid testing in the past 10 years have been summarized. Fluorescence enhancement and red shift can occur when the G-rich sequence gets close to Ag NCs or the complementary DNA strand hybridizes with Ag NCs tail strand, which can be used to identify the nucleic acid. Ag NCs with the abasic site in DNA duplex can distinguish mutant genes such as cancer suppression gene p53. Ag NCs with auxiliary DNA can be used to detect miR-21, miR-16-5p, miR-19b-3p, and miR-141. Cu NCs/Cu NPs can recognize miRNA-155, miR-21, and miR-let-7d without auxiliary DNA. Au NCs can identify H1N1 gene fragments by fluorescence quenching caused by proximity to the G-rich sequence. Besides, Au NCs can recognize miRNA-21 and let-7a. SsDNA MNCs adsorbed on the surface of GO and CNPs oxide can be used to identify HBV and HIV gene fragments. The addition of enzymes or auxiliary amplification technologies is a popular way to improve probe sensitivity. Ag NCs combined with TAIEA has the best performance and can obtain LOD as low as aM for miRNA.

In this work, an environmentally-friendly and versatile nanobeacon was constructed by utilizing DNA-templated copper nanoparticles (CuNPs) as fluorescence signal source. As the key component of the nanobeacon, a hairpin DNA was designed to contain four segments: two segments for CuNPs template sequence, a target recognition segment and a blocking segment. At room temperature, the target recognition segment partly hybridizes with the blocking segment and thus prohibits the formation of double stranded DNA template, so that no CuNPs can be generated on the hairpin DNA. While a target is introduced, the specific binding of target with recognition sequence triggers off the conformational transformation of the hairpin DNA, which contributes to the formation of the CuNPs template. As a result, the in-situ generation of CuNPs gives birth to the fluorescence signal readout that can be used to identify the target. By reasonably varying the recognition sequence within hairpin DNA, a series of nanobeacons in response to corresponding targets, such as DNA, microRNA, thrombin, and ATP, were put forward with satisfactory sensitivity and selectivity. Moreover, this nanobeacon was also integrated with the strategy of enzyme-assisted target-recycling to realize signal amplification and ultrasensitive detection, which further demonstrated the versatility of the nanobeacon. This novel nanobeacon is expected to be a promising alternative to classical dye-labeled molecular beacon and provide new perspective on ultrasensitive fluorescence sensing.

With the rapid development of nanotechnology, new types of fluorescent nanomaterials (FNMs) have been springing up in the past two decades. The nanometer scale endows FNMs with unique optical properties which play a critical role in their applications in bioimaging and fluorescence-dependent detections. However, since low selectivity as well as low photoluminescence efficiency of fluorescent nanomaterials hinders their applications in imaging and detection to some extent, scientists are still in search of synthesizing new FNMs with better properties. In this review, a variety of fluorescent nanoparticles are summarized including semiconductor quantum dots, carbon dots, carbon nanoparticles, carbon nanotubes, graphene-based nanomaterials, noble metal nanoparticles, silica nanoparticles, phosphors and organic frameworks. We highlight the recent advances of the latest developments in the synthesis of FNMs and their applications in the biomedical field in recent years. Furthermore, the main theories, methods, and limitations of the synthesis and applications of FNMs have been reviewed and discussed. In addition, challenges in synthesis and biomedical applications are systematically summarized as well. The future directions and perspectives of FNMs in clinical applications are also presented.

Ultrasensitive biosensors have become a necessity in the world of scientific research, and several signal enhancement strategies have been employed to attain exceptionally low detection limits. Nanotechnology turns out to be a strong contender for signal amplification, as they can be employed as platform modifiers, catalysts, carriers or labels. Here, we have described the most recent advancements in the utilization of nanomaterials as signal amplification components in aptamer-based electrochemical biosensors. We have briefly reviewed the methods that utilized nanomaterials, namely gold and carbon, as well as nanocomposites such as: graphene, carbon nanotubes, quantum dots, and metal-organic frameworks.

With the active development of mobile devices, a variety of ultra-small, high-definition, and open platform-based cameras are being mass-produced. In this paper, we established an emulation system to verify the bio-imaging performance of the bulky and expensive high-performance cameras and various smartphone cameras that have been used in bio-imaging devices. In the proposed system, the linearity of the brightness gradient change of four types of cameras was compared and analyzed. Based on these results, three cameras were selected in order of excellent linearity, and gel image analysis results were compared.

Tuberculosis is one of devastating infectious diseases in the world, and early diagnosis and treatment can help overcome this global burden. In this work, a new detection platform combining smartphone-assisted fluorescent analysis and highly sensitive fluorescent copper nanoprobes (CuNPs) in a specific nucleic acid amplification test (NAAT) for the diagnosis of tuberculosis (TB) was demonstrated and validated using clinical samples. To enhance the precision and accuracy of detection, polymerase chain reaction (PCR), padlock probe (PLP) ligation, and rolling circle amplification (RCA) were combined. Long poly(thymine) (polyT) single-stranded DNA was synthesized through RCA, and polyT-CuNPs were formed by adding copper(II) ions and sodium ascorbate as reducing agents; subsequently, the results were visualized through the excitation from a UV transilluminator and quantified with just a smartphone. After optimization, this proposed platform was validated by testing 18 residual DNA samples after TB PCR, including 8 TB-negative and 10 TB-positive samples, and exhibited a detection limit of 5 fg/μL. The findings indicate the potential of this platform for practical application, where it can be combined with a smartphone for image analysis to achieve accurate on-site detection of TB, especially in resource-limited settings.

Herein, we synthesized snowflake-like DNA crystals (SDC) via hybridization chain reaction and used it for the first time in the synthesis of copper nanoclusters with enhanced fluorescence. Atomic force microscopy (AFM) and laser confocal microscopy characterization confirmed that SDC/CuNCs are self-assembled successfully on SDC. Aggregation induced emission allows SDC/CuNCs to exhibit better stability and stronger emission intensity. Thus, we developed the "turn-on" label-free fluorescence detection method of actin based on SDC/CuNCs which offer simplicity, low cost, good selectivity, and high sensitivity. The detection limit was determined to be 0.0124 μg mL^-1, which was an order of magnitude lower than that of reported fluorescent methods (0.12 μg mL^-1). Compared with previous method, the linear range is also much wider. We also performed standard recovery experiments in actual samples for evaluating the practicality of this strategy and proved that the capability of the proposed approach for the determination of actin is feasible and the interference from complex biological samples is negligible. These results indicate that SDC/CuNCs are expected to play a more important role in the field of biosensors.

In this work, an ultrasensitive and turn-on sensor for homogeneous Hg2+ detection has been constructed based on a target-triggered isothermal cycling reaction and rapid label-free signal output with dsDNA-templated copper nanoparticles (CuNPs). As the key component of the sensor, a hairpin DNA without any labels was designed to contain different functional sequence segments and to resist digestion by exonuclease due to the protruding 3'-terminus. In the presence of Hg2+, the formation of a T-Hg2+-T structure turned the protruding 3'-terminus of the hairpin DNA to a blunt end that could be efficiently digested by Exo III, accompanied by Hg2+ release, followed by another digestion cycle. Hence, the Hg2+-triggered isothermal cycling reaction accumulated numerous dsDNA templates that facilitated fluorescent CuNP generation and finally output an amplified signal used to identify the target. This protocol is capable of Hg2+ sensing in a concentration range of 5 orders of magnitude with a detection limit down to 3.9 pM. The as-constructed sensor also revealed high selectivity, as well as satisfactory results in recovery experiments of Hg2+ detection in real water samples.

The real motivation in the present work is to tune the synthesis variables that can result in a highly fluorescent and stable DNA copper nanocluster (CuNC) and also to understand the intricate mechanism behind this process. Here, carefully optimized concentrations of various reactants enabled the creation of a DNA-encapsulated CuNC for AT-DNA, displaying a size of <1.0 nm as confirmed by transmission electron microscopy and dynamic light scattering. The extremely small size of the AT-DNACuNC supports the discrete electronic transitions, also characterized by an exceptionally strong negative circular dichroism (CD) band around 350 nm, whose intensity is well correlated with the observed strong fluorescence emission intensity. This remarkably strong CD can open new applications in the detection and quantification of a specific DNACuNC. Further, time-dependent fluorescence analysis suggested stronger photostabilization of these DNACuNCs. The simulation study, based on Cu ion distribution, explained how AT-DNA is a better candidate for NC formation than GC-DNA. In conclusion, the better-tuned synthesis procedure has resulted in a highly compact, well-defined three-dimensional conformation that promotes a more favorable microenvironment to sequester a DNA-based CuNC with high brightness and outstanding photostability.