Magnetic Bead/Gold Nanoparticle Double‐Labeled Primers for Electrochemical Detection of Isothermal Amplified Leishmania DNA

A novel methodology for the isothermal amplification of Leishmania DNA using labeled primers combined with the advantages of magnetic purification/preconcentration and the use of gold nanoparticle (AuNP) tags for the sensitive electrochemical detection of such amplified DNA is developed. Primers labeled with AuNPs and magnetic beads (MBs) are used for the first time for the isothermal amplification reaction, being the amplified product ready for the electrochemical detection. The electrocatalytic activity of the AuNP tags toward the hydrogen evolution reaction allows the rapid quantification of the DNA on screen‐printed carbon electrodes. Amplified products from the blood of dogs with Leishmania (positive samples) are discriminated from those of healthy dogs (blank samples). Quantitative studies demonstrate that the optimized method allows us to detect less than one parasite per microliter of blood (8 × 10^?3 parasites in the isothermal amplification reaction). This pioneering approach is much more sensitive than traditional methods based on real‐time polymerase chain reaction (PCR), and is also more rapid, cheap, and user‐friendly.     

Modulation of DNA Polymerases with Gold Nanoparticles and their Applications in Hot‐Start PCR

A new gold‐nanoparticle (AuNP)‐based strategy to dynamically modulate the activity of DNA polymerases and realize a hot‐start (HS)‐like effect in the polymerase chain reaction (PCR) is reported, which effectively prevents unwanted nonspecific amplification and improves the performance of PCRs. A high‐fidelity Pfu DNA polymerase is employed as the model system. Interestingly, AuNPs inactivate the polymerase activity of Pfu at low temperature, thus resembling an antibody‐based HS PCR. This inhibition effect of AuNPs is demonstrated for the preamplification polymerization activity of the PCR, which largely suppresses nonspecific amplification at temperatures between 30 and 60 °C and leads to highly specific and sensitive PCR amplification with Pfu. Significantly, the fidelity of Pfu is not sacrificed in the presence of AuNPs. Therefore, this AuNP‐based HS strategy provides a straightforward and potentially versatile approach to realize high‐performance PCR amplification.     

Site‐Specific SERS Assay for Survivin Protein Dimer: From Ensemble Experiments to Correlative Single‐Particle Imaging

An assay for Survivin, a small dimeric protein which functions as modulator of apoptosis and cell division and serves as a promising diagnostic biomarker for different types of cancer, is presented. The assay is based on switching on surface‐enhanced Raman scattering (SERS) upon incubation of the Survivin protein dimer with Raman reporter‐labeled gold nanoparticles (AuNP). Site‐specificity is achieved by complexation of nickel‐chelated N‐nitrilo‐triacetic acid (Ni‐NTA) anchors on the particle surface by multiple histidines (His₆‐tag) attached to each C‐terminus of the centrosymmetric protein dimer. Correlative single‐particle analysis using light sheet laser microscopy enables the simultaneous observation of both elastic and inelastic light scattering from the same sample volume. Thereby, the SERS‐inactive AuNP‐protein monomers can be directly discriminated from the SERS‐active AuNP‐protein dimers/oligomers. This information, i.e. the percentage of SERS‐active AuNP in colloidal suspension, is not accessible from conventional SERS experiments due to ensemble averaging. The presented correlative single‐particle approach paves the way for quantitative site‐specific SERS assays in which site‐specific protein recognition by small chemical and in particular supramolecular ligands can be tested.     

Split‐GFP: SERS Enhancers in Plasmonic Nanocluster Probes

The assembly of plasmonic metal nanoparticles into hot spot surface‐enhanced Raman scattering (SERS) nanocluster probes is a powerful, yet challenging approach for ultrasensitive biosensing. Scaffolding strategies based on self‐complementary peptides and proteins are of increasing interest for these assemblies, but the electronic and the photonic properties of such hybrid nanoclusters remain difficult to predict and optimize. Here, split‐green fluorescence protein (sGFP) fragments are used as molecular glue and the GFP chromophore is used as a Raman reporter to assemble a variety of gold nanoparticle (AuNP) clusters and explore their plasmonic properties by numerical modeling. It is shown that GFP seeding of plasmonic nanogaps in AuNP/GFP hybrid nanoclusters increases near‐field dipolar couplings between AuNPs and provides SERS enhancement factors above 10⁸. Among the different nanoclusters studied, AuNP/GFP chains allow near‐infrared SERS detection of the GFP chromophore imidazolinone/exocyclic C?C vibrational mode with theoretical enhancement factors of 10⁸–10⁹. For larger AuNP/GFP assemblies, the presence of non‐GFP seeded nanogaps between tightly packed nanoparticles reduces near‐field enhancements at Raman active hot spots, indicating that excessive clustering can decrease SERS amplifications. This study provides rationales to optimize the controlled assembly of hot spot SERS nanoprobes for remote biosensing using Raman reporters that act as molecular glue between plasmonic nanoparticles.     

Nanozyme-Based Colorimetric SARS-CoV-2 Nucleic Acid Detection by Naked Eye

Fluorescence-based PCR and other amplification methods have been used for SARS-CoV-2 diagnostics, however, it requires costly fluorescence detectors and probes limiting deploying large-scale screening. Here, a cut-price colorimetric method for SARS-CoV-2 RNA detection by iron manganese silicate nanozyme (IMSN) is established. IMSN catalyzes the oxidation of chromogenic substrates by its peroxidase (POD)-like activity, which is effectively inhibited by pyrophosphate ions (PPi). Due to the large number of PPi generated by amplification processes, SARS-CoV-2 RNA can be detected by a colorimetric readout visible to the naked eye, with the detection limit of 240 copies mL⁻¹. This conceptually new method has been successfully applied to correctly distinguish positive and negative oropharyngeal swab samples of COVID-19. Colorimetric assay provides a low-cost and instrumental-free solution for nucleic acid detection, which holds great potential for facilitating virus surveillance.     

Changes in telomerase activity after irradiation of human peripheral blood mononuclear cells (PBMC) in vitro

Telomerase activity (TA) has been shown to be up-regulated by ionising radiation in immortal hematopoietic cell lines. The purpose of the present experiments was to test whether ionising radiation may regulate TA in normal human peripheral blood mononuclear cells (PBMC). A real-time PCR assay was established to quantify TA detected by the telomeric repeat amplification protocol. TA of PBMCs isolated from young healthy donors was highly increased by stimulation with phytohemagglutinin (PHA) for 72 h. Irradiation of PHA-stimulated PBMCs with 2-10 Gy of X rays showed up-regulated TA 4 h after irradiation with an enhancement at least as strong as for the human TK6 lymphoblastoid cell line. The present results show that TA is up-regulated by irradiation not only in immortal cell lines but also in mitogen-stimulated PBMCs. This supports a possible role for telomerase in the cellular radiation response.     

In vivo Gold Nanoparticle Delivery of Peptide Vaccine Induces Anti‐Tumor Immune Response in Prophylactic and Therapeutic Tumor Models

Gold nanoparticles (AuNPs) are promising vehicles for cancer immunotherapy, with demonstrated efficacy in immune delivery and innate cell stimulation. Nevertheless, their potential has yet to be assessed in the in vivo application of peptide cancer vaccines. In this study, it is hypothesized that the immune distribution and adjuvant qualities of AuNPs could be leveraged to facilitate delivery of the ovalbumin (OVA) peptide antigen and the CpG adjuvant and enhance their therapeutic effect in a B16‐OVA tumor model. AuNP delivery of OVA (AuNP‐OVA) and of CpG (AuNP‐CpG) enhanced the efficacy of both agents and induced strong antigen‐specific responses. In addition, it is found that AuNP‐OVA delivery alone, without CpG, is sufficient to promote significant antigen‐specific responses, leading to subsequent anti‐tumor activity and prolonged survival in both prophylactic and therapeutic in vivo tumor models. This enhanced therapeutic efficacy is likely due to the adjuvant effect of peptide coated AuNPs, as they induce inflammatory cytokine release when cultured with bone marrow dendritic cells. Overall, AuNP‐mediated OVA peptide delivery can produce significant therapeutic benefits without the need of adjuvant, indicating that AuNPs are effective peptide vaccine carriers with the potential to permit the use of lower and safer adjuvant doses during vaccination.     

TGF‐β1 Conjugated to Gold Nanoparticles Results in Protein Conformational Changes and Attenuates the Biological Function

Gold nanoparticles (AuNPs) are widely used as carriers or therapeutic agents due to their great biocompatibility and unique physical properties. Transforming growth factor‐beta 1 (TGF‐β1), a member of the cysteine‐knot structural superfamily, plays a pivotal role in many diseases and is known as an immunosuppressive agent that attenuates immune response resulting in tumor growth. The results reported herein reflect strong interactions between TGF‐β1 and the surface of AuNPs when incubated with serum‐containing medium, and demonstrate a time‐ and dose‐dependent pattern. Compared with other serum proteins that can also bind to the AuNP surface, AuNP–TGFβ1 conjugate is a thermodynamically favored compound. Epithelial cells undergo epithelial–mesenchymal transition (EMT) upon treatment with TGF‐β1; however, treatment with AuNPs reverses this effect, as detected by cell morphology and expression levels of EMT markers. TGF‐β1 is found to bind to AuNPs through S–Au bonds by X‐ray photoelectron spectroscopy. Fourier transform infrared spectroscopy is employed to analyze the conformational changes of TGF‐β1 on the surface of AuNPs. The results indicate that TGF‐β1 undergoes significant conformational changes at both secondary and tertiary structural levels after conjugation to the AuNP surface, which results in the deactivation of TGF‐β1 protein. An in vivo experiment also shows that addition of AuNPs attenuates the growth of TGF‐β1‐secreting murine bladder tumor 2 cells in syngeneic C3H/HeN mice, but not in immunocompromised NOD‐SCID mice, and this is associated with an increase in the number of tumor‐infiltrating CD4⁺ and CD8⁺ T lymphocytes and a decrease in the number of intrasplenic Foxp3(+) lymphocytes. The findings demonstrate that AuNPs may be a promising agent for modulating tumor immunity through inhibiting immunosuppressive TGF‐β1 signaling.     

A Centimeter‐Scale Inorganic Nanoparticle Superlattice Monolayer with Non‐Close‐Packing and its High Performance in Memory Devices

Due to the near‐field coupling effect, non‐close‐packed nanoparticle (NP) assemblies with tunable interparticle distance (d) attract great attention and show huge potential applications in various functional devices, e.g., organic nano‐floating‐gate memory (NFGM) devices. Unfortunately, the fabrication of device‐scale non‐close‐packed 2D NPs material still remains a challenge, limiting its practical applications. Here, a facile yet robust “rapid liquid–liquid interface assembly” strategy is reported to generate a non‐close‐packed AuNP superlattice monolayer (SM) on a centimeter scale for high‐performance pentacene‐based NFGM. The d and hence the surface plasmon resonance spectra of SM can be tailored by adjusting the molecular weight of tethered polymers. Precise control over the d value allows the successful fabrication of photosensitive NFGM devices with highly tunable performances from short‐term memory to nonvolatile data storage. The best performing nonvolatile memory device shows remarkable 8‐level (3‐bit) storage and a memory ratio over 10⁵ even after 10 years compared with traditional devices with a AuNP amorphous monolayer. This work provides a new opportunity to obtain large area 2D NPs materials with non‐close‐packed structure, which is significantly meaningful to microelectronic, photovoltaics devices, and biochemical sensors.     

Magnetic Plasmon Networks Programmed by Molecular Self‐Assembly

Nanoscale manipulation of magnetic fields has been a long‐term pursuit in plasmonics and metamaterials, as it can enable a range of appealing optical properties, such as high‐sensitivity circular dichroism, directional scattering, and low‐refractive‐index materials. Inspired by the natural magnetism of aromatic molecules, the cyclic ring cluster of plasmonic nanoparticles (NPs) has been suggested as a promising architecture with induced unnatural magnetism, especially at visible frequencies. However, it remains challenging to assemble plasmonic NPs into complex networks exhibiting strong visible magnetism. Here, a DNA‐origami‐based strategy is introduced to realize molecular self‐assembly of NPs forming complex magnetic architectures, exhibiting emergent properties including anti‐ferromagnetism, purely magnetic‐based Fano resonances, and magnetic surface plasmon polaritons. The basic building block, a gold NP (AuNP) ring consisting of six AuNP seeds, is arranged on a DNA origami frame with nanometer precision. The subsequent hierarchical assembly of the AuNP rings leads to the formation of higher‐order networks of clusters and polymeric chains. Strong emergent plasmonic properties are induced by in situ growth of silver upon the AuNP seeds. This work may facilitate the development of a tunable and scalable DNA‐based strategy for the assembly of optical magnetic circuitry, as well as plasmonic metamaterials with high fidelity.     

Carbon Nanotubes Multifunctionalized by Rolling Circle Amplification and Their Application for Highly Sensitive Detection of Cancer Markers

There are still challenges for the development of multifunctional carbon nanotubes (CNTs). Here, a multiwalled carbon nanotube (MWCNT)‐based rolling circle amplification system (CRCAS) is reported which allows in situ rolling circle replication of DNA primer on the surface of MWCNTs to create a long single‐strand DNA (ssDNA) where a large number of nanoparticles or proteins could be loaded, forming a nano‐biohybridized 3D structure with a powerful signal amplification ability. In this strategy, the binding ability of proteins, hybridization, replication ability of DNA, and the catalytical ability of enzymes are integrated on a single carbon nanotube. The CRCAS is then used to develop colorimetric and chemiluminescent assays for the highly sensitive and specific detection of cancer protein markers, alpha‐fetoprotein (AFP) and prostate specific antigen (PSA). The colorimetric CRCAS assay is 4000 times more sensitive than a conventional enzyme‐linked immunosorbent assay (ELISA), and its concentration range is 10 000 times wider. Control experiments show that as low as 10 pg mL^?1 AFP or PSA could be detected even in the presence of interfering protein markers with a more than 10⁵‐fold greater concentration in the sample, demonstrating the high specificity of the CRCAS assay. The limit of detection of the chemiluminescent CRCAS assays for AFP and PSA are 5 fg mL^?1 (70 aM) and 10 fg mL^?1 (0.29 fM), respectively, indicating that the sensitivity is much higher than that of the colorimetric CRCAS assay. Importantly, CRCAS works well with real biological samples.     

Interfacial Assembly of Signal Amplified Multienzymes and Biorecognized Antibody into Proteinosome for an Ultrasensitive Immunoassay

Enzyme as signal tag has been widely employed in colorimetric immunoassays for decades. Nevertheless, it remains a great challenge to substantially improve the detection sensitivity of enzyme‐based immunoassays, which inhibits further critical applications. To circumvent this confinement, a multifunctional self‐assembled proteinosome based on the integration of signal amplification elements (enzyme) and biorecognition unit (antibody) is proposed for fabricating an immunoassay strategy with significantly enhanced sensitivity. Owing to the self‐assembly technique, this proteinosome not only efficiently loads abundant enzymes to possess high catalytic activity, but also enhances enzymatic stability and maintains recognition ability of antibody. Using imidacloprid as a model target, the proteinosome‐based immunoassay reaches a limit of detection down to the picogram mL^?1 level, which is 150‐fold lower than that of conventional enzyme‐linked immunosorbent assay. This method provides a versatile approach for constructing spherical proteinosome as a recognizer and amplifier for profiling a broad range of target antigen.     

Self‐Assembly of Poly‐Adenine‐Tailed CpG Oligonucleotide‐Gold Nanoparticle Nanoconjugates with Immunostimulatory Activity

Synthetic unmethylated cytosine–guanine (CpG) oligodeoxynucleotides (CpG ODNs) possess high immunostimulatory activity and have been widely used as a therapeutic tool for various diseases including infection, allergies, and cancer. A variety of nanocarriers have been developed for intracellular delivery of CpG ODNs that are otherwise nonpermeable through the cellular membrane. For example, previous studies showed that gold nanoparticles (AuNPs) could efficiently deliver synthetic thiolated CpG ODNs into cultured cells and induce expression of proinflammatory cytokines. Nevertheless, the necessity of using thiolated CpG ODNs for the modification of AuNPs inevitably complicates the synthesis of the nanoconjugates and increases the cost. A new approach is demonstrated for facile assembly of AuNP‐CpG nanoconjugates for cost‐effective drug delivery. It is found that non‐thiolated, diblock ODNs containing a CpG motif and a poly‐adenine (polyA) tail can readily self‐assemble on the surface of AuNPs with controllable and tunable density. Such nanoconjugates are efficiently delivered into RAW264.7 cells and induce immune response in a Toll‐like receptor 9 (TLR9)‐dependent manner. Under optimal conditions, polyA‐CpG‐AuNPs show significantly higher immunostimulatory activity than their thiolated counterpart. In addition, the immunostimulatory activity of CpG‐AuNPs can be modulated by varying the length of the polyA tail. In vivo induction of immune responses in mice is demonstrated by using polyA‐tailed CpG‐AuNP nanoconjugates.     

The Role of Temperature and Lipid Charge on Intake/Uptake of Cationic Gold Nanoparticles into Lipid Bilayers

Understanding the molecular mechanisms governing nanoparticle–membrane interactions is of prime importance for drug delivery and biomedical applications. Neutron reflectometry (NR) experiments are combined with atomistic and coarse‐grained molecular dynamics (MD) simulations to study the interaction between cationic gold nanoparticles (AuNPs) and model lipid membranes composed of a mixture of zwitterionic di‐stearoyl‐phosphatidylcholine (DSPC) and anionic di‐stearoyl‐phosphatidylglycerol (DSPG). MD simulations show that the interaction between AuNPs and a pure DSPC lipid bilayer is modulated by a free energy barrier. This can be overcome by increasing temperature, which promotes an irreversible AuNP incorporation into the lipid bilayer. NR experiments confirm the encapsulation of the AuNPs within the lipid bilayer at temperatures around 55 °C. In contrast, the AuNP adsorption is weak and impaired by heating for a DSPC–DSPG (3:1) lipid bilayer. These results demonstrate that both the lipid charge and the temperature play pivotal roles in AuNP–membrane interactions. Furthermore, NR experiments indicate that the (negative) DSPG lipids are associated with lipid extraction upon AuNP adsorption, which is confirmed by coarse‐grained MD simulations as a lipid‐crawling effect driving further AuNP aggregation. Overall, the obtained detailed molecular view of the interaction mechanisms sheds light on AuNP incorporation and membrane destabilization.     

Regulation of cell fate by near infrared-responsive telomerase activity

Here we develop a strategy using near infrared (NIR) modulation of telomerase activity based on gold nanocage@smart polymer system. Using this biocompatible design, we can regulate cellular behavior. This system has been used in vivo by taking advantages of NIR. This is the first example for optical modulation of telomerase activity in living cells and tissues.

     

Colorimetric Detection of Hg2+ Based on the Growth of Aptamer‐Coated AuNPs: The Effect of Prolonging Aptamer Strands

Herein, a versatile and sensitive colorimetric sensor for Hg²⁺ based on aptamer–target specific binding and target‐mediated growth of AuNPs is reported. The 15 T bases are first designed to detect Hg²⁺ through T–Hg²⁺–T coordination. Aptamer–target binding results in the desorption of the aptamer from AuNP surface, the remaining aptamers adsorbed on AuNP surface trigger the growth of AuNPs with morphologically varied nanostructures, and then different colored solutions are formed. On this occasion, the limit of detection (LOD) of 9.6 × 10^?9m is obtained. The other two aptamer strands (25‐ and 59‐mer) are designed by increasing A bases on either side and both sides of 15 T, respectively. The interaction of the binding domain and Hg²⁺ makes desorption of 15 T from AuNP surface, whereas excess bases not committed to the binding domain still adsorbed on AuNP surface. These excess bases control the growth of AuNPs, and enhance the sensitivity. The LODs are 4.05 and 3 × 10^?9m for 25‐ and 59‐mer aptamers, respectively. In addition, the 59‐mer aptamer system is applied to identify Hg²⁺ in real river samples, the LOD of 6.2 × 10^?9m is obtained.     

Highly Sensitive Detection of Protein Biomarkers with Organic Electrochemical Transistors

The analysis of protein biomarkers is of great importance in the diagnosis of diseases. Although many convenient and low‐cost electrochemical approaches have been extensively investigated, they are not sensitive enough in the detection of protein biomarkers with low concentrations in physiological environments. Here, this study reports a novel organic‐electrochemical‐transistor‐based biosensor that can successfully detect cancer protein biomarkers with ultrahigh sensitivity. The devices are operated by detecting electrochemical activity on gate electrodes, which is dependent on the concentrations of proteins labeled with catalytic nanoprobes. The protein sensors can specifically detect a cancer biomarker, human epidermal growth factor receptor 2, down to the concentration of 10^?14 g mL^?1, which is several orders of magnitude lower than the detection limits of previously reported electrochemical approaches. Moreover, the devices can successfully differentiate breast cancer cells from normal cells at various concentrations. The ultrahigh sensitivity of the protein sensors is attributed to the inherent amplification function of the organic electrochemical transistors. This work paves a way for developing highly sensitive and low‐cost biosensors for the detection of various protein biomarkers in clinical analysis in the future.     

Nanoparticle Linker‐Controlled Molecular Wire Devices Based on Double Molecular Monolayers

Highly conductive molecular wires are an important component for realizing molecular electronic devices and have to be explored in terms of interactions between molecules and electrodes in their molecular junctions. Here, new molecular wire junctions are reported to enhance charge transport through gold nanoparticle (AuNP)‐linked double self‐assembled monolayers (SAMs) of cobalt (II) bis‐terpyridine molecules (e.g., Co(II)(tpyphS)₂). Electrical characteristics of the double‐SAM devices are explored in terms of the existence of AuNP. The AuNP linker in the Co(II)(tpyphS)₂–AuNP–Co(II)(tpyphS)₂ junction acts as an electronic contact that is transparent to electrons. The weak temperature dependency of the AuNP‐linked molecular junctions strongly indicates sequential tunneling conduction through the highest occupied molecular orbitals (HOMOs) of Co(II)(tpyphS)₂ molecules. The electrochemical characteristics of the AuNP–Co(II)(tpyphS)₂ SAMs reveal fast electron transfer through molecules linked by AuNP. Density functional theory calculations reveal that the molecular HOMO levels are dominantly affected by the formation of junctions. The intermolecular charge transport, controlled by the AuNP linker, can provide a rational design for molecular connection that achieves a reliable electrical connectivity of molecular electronic components for construction of molecular electronic circuits.     

Chirality‐Selective Photoluminescence Enhancement of ssDNA‐Wrapped Single‐Walled Carbon Nanotubes Modified with Gold Nanoparticles

In this work, a convenient method to enhance the photoluminescence (PL) of single‐walled carbon nanotubes (SWNTs) in aqueous solutions is provided. Dispersing by single‐stranded DNA (ssDNA) and modifying with gold nanoparticles (AuNPs), about tenfold PL enhancement of the SWNTs is observed. More importantly, the selective PL enhancement is achieved for some particular chiralities of interest over all other chiralities, by using certain specific ssDNA sequences that are reported to recognize these particular chiralities. By forming AuNP–DNA–SWNT nanohybrids, ssDNA serves as superior molecular spacers that on one hand protect SWNT from direct contacting with AuNP and causing PL quench, and on the other hand attract the AuNP in close proximity to the SWNT to enhance its PL. This PL enhancement method can be utilized for the PL analysis of SWNTs in aqueous solutions, for biomedical imaging, and may serve as a prescreening method for the recognition and separation of single chirality SWNTs by ssDNA.     

Unusual switchable peroxidase-mimicking nanozyme for the determination of proteolytic biomarker

Detection of enzyme biomarkers originating from either bio-fluids or contaminating microorganisms is of utmost importance in clinical diagnostics and food safety. Herein, we present a simple, low-cost and easy-to-use sensing approach based on the switchable peroxidase-mimicking activity of plasmonic gold nanoparticles (AuNPs) that can catalyse for the oxidation of 3,3’,5’5-tetramethylbenzidine (TMB) for the determination of protease enzyme. The AuNP surface is modified with casein, showing dual functionalities. The first function of the coating molecule is to suppress the intrinsic peroxidase-mimicking activity of AuNPs by up to 77.1%, due to surface shielding effects. Secondly, casein also functions as recognition sites for the enzyme biomarker. In the presence of protease, the enzyme binds to and catalyses the degradation of the coating layer on the AuNP surface, resulting in the recovery of peroxidase-mimicking activity. This is shown visually in the development of a blue colored product (oxidised TMB) or spectroscopically as an increase in absorbance at 370 and 650 nm. This mechanism allows for the detection of protease at 44 ng·mL⁻¹ in 90 min. The nanosensor circumvents issues associated with current methods of detection in terms of ease of use, compatibility with point-of-care testing, low-cost production and short analysis time. The sensing approach has also been applied for the detection of protease spiked in ultra-heat treated (UHT) milk and synthetic human urine samples at a limit of detection of 490 and 176 ng·mL⁻¹, respectively, showing great potential in clinical diagnostics, food safety and quality control.