Copper (II)-catalyzed polydopamine mediated photothermal sensors for visual quantitative point-of-care testing.

BACKGROUND: Temperature sensing is commonly used in point-of-care (POC) detection technologies, yet the portability and convenience of use are frequently compromised by the complexity of thermosensitive processes and signal transduction. Especially, multi-step target recognition reactions and temperature measurement in the reaction vessel present challenges in terms of stability and integration of detection devices. To further combine photothermal reaction and signal readout in one assay, these two processes enable to be integrated into miniaturized microfluidic chips, thereby facilitating photothermal sensing and achieving a simple visual temperature sensing as POC detection. RESULTS: A copper ion (Cu2+)-catalyzed photothermal sensing system integrated onto a microfluidic distance-based analytical device (µDAD), enabling the visual, portable, and sensitive quantitative detection of multiple targets, including ascorbic acid, glutathione, and alkaline phosphatase (ALP). The polydopamine nanoparticles (PDA NPs) were synthesized by the regulation of free Cu2+ through redox or coordination reactions, facilitating the transduction of distinct photothermal response signals and providing the versatile Cu2+-responsive sensing systems. Promoted by integration with a photothermal µDAD, the system combines PDA's photothermal responsiveness and thermosensitive gas production of ammonium bicarbonate for improved sensitivity of ALP detection, reaching the detection limit of 9.1 mU/L. The system has successfully achieved on-chip detection of ALP with superior anti-interference capability and recoveries ranging from 96.8 % to 104.7 %, alongside relative standard deviations below 8.0 %. SIGNIFICANCE AND NOVELTY: The µDAD design accommodated both the photothermal reaction of PDA NPs and thermosensitive gas production reaction, achieving the rapid sensing of visual distance signals. The µDAD-based Cu2+-catalyzed photothermal sensing system holds substantial potential for applications in biochemical analysis and clinical diagnostics, underscored by the versatile Cu2+ regulation mechanism for a broad spectrum of biomarkers.

Rapid and sensitive detection of SARS-CoV-2 based on a phage-displayed scFv antibody fusion with alkaline phosphatase and NanoLuc luciferase.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the subsequent pandemic have led to devastating public health and economic losses. The development of highly sensitive, rapid and inexpensive methods to detect and monitor coronaviruses is essential for family diagnosis, preventing infections, choosing treatments and programs and laying the technical groundwork for viral diagnosis. This study established one-step immunoassays for rapid and sensitive detection of SARS-CoV-2 by using a single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) or NanoLuc (NLuc) luciferase. First, a high-affinity scFv antibody specific to the SARS-CoV-2 nucleocapsid (N) protein was screened from hybridoma cells-derived and phage-displayed library. Next, prokaryotic expression of the scFv-AP and scFv-NLuc fusion proteins were induced, leading to excellent antibody binding properties and enzyme catalytic activities. The scFv-AP fusion had a detection limit of 3 pmol per assay and was used to produce eye-readable biosensor readouts. Moreover, the scFv-NLuc protein was applied in a highly sensitive luminescence immunoassay, achieving a detection limit lower than 0.1 pmol per assay. Therefore, the scFv-AP and scFv-NLuc fusion proteins can be applied for the rapid and simple diagnosis of SARS-CoV-2 to safeguard human health and provide guidance for the detection of other pathogenic viruses.

Alkaline Phosphatase-Instructed Peptide Assemblies for Imaging and Therapeutic Applications.

Self-assembly, a powerful strategy for constructing highly stable and well-ordered supramolecular structures, widely exists in nature and in living systems. Peptides are frequently used as building blocks in the self-assembly process due to their advantageous characteristics, such as ease of synthesis, tunable mechanical stability, good biosafety, and biodegradability. Among the initiators for peptide self-assembly, enzymes are excellent candidates for guiding this process under mild reaction conditions. As a crucial and commonly used biomarker, alkaline phosphatase (ALP) cleaves phosphate groups, triggering a hydrophilicity-to-hydrophobicity transformation that induces peptide self-assembly. In recent years, ALP-instructed peptide self-assembly has made breakthroughs in biological imaging and therapy, inspiring the development of self-assembly biomaterials for diagnosis and therapeutics. In this review, we highlight the most recent advancements in ALP-instructed peptide assemblies and provide perspectives on their potential impact. Finally, we briefly discuss the ongoing challenges for future research in this field.

Guide DNA dephosphorylation-modulated Pyrococcus furiosus Argonaute fluorescence biosensor for the detection of alkaline phosphatase and aflatoxins B1.

Foodborne hazardous factors pose a significant risk to public health, emphasizing the need for the development of sensitive and user-friendly detection strategies to effectively manage and control these risks in the food supply chain. Pyrococcus furiosus argonaute (PfAgo)-based biosensing approaches have been extensively explored due to its built-in signal amplification. However, the property that PfAgo is a DNA-guided DNA endonuclease has enabled almost all the existing PfAgo-based reports to be used for the detection of nucleic acids. To lend PfAgo toolbox to extended non-nucleic acid detection, we systematically investigated the mechanism characteristic of PfAgo' preference for guide DNA (gDNA) and proposed a gDNA dephosphorylation-modulated PfAgo sensor for the detection of non-nucleic acid targets. Our results indicated that PfAgo exhibits preference for 5'-phosphorylated gDNA at a specific ratio of PfAgo to gDNA concentration. Leveraging this PfAgo' preference and the dephosphorylation activity of alkaline phosphatase (ALP), ALP could be detected as low as 2.7 U/L. Furthermore, the PfAgo was coupled with immunolabelled ALP to develop a PfAgo-based fluorescence immunosensor, which achieves aflatoxins B1 detection with a detection limit of 29.89 pg/mL and exhibits satisfactory recoveries in wheat and maize samples. The developed method broadens the application scope of PfAgo toolbox, and provides a simple, sensitive, and universal detection platform for a variety targets.

Nanoflower Microreactor Based Versatile Enhancer for Recognition Cofactor-Dependent Enzyme Biocatalysis toward Saxitoxin Detection.

Investigating organic carriers' utilization efficiency and bioactivity within organic-inorganic hybrid nanoflowers is critical to constructing sensitive immunosensors. Nevertheless, the sensitivity of immunosensors is interactively regulated by different classes of biomolecules such as antibodies and enzymes. In this work, we introduced a new alkaline phosphatase-antibody-CaHPO4 hybrid nanoflowers (AAHNFs) microreactor based colorimetric immunoprobe. This system integrates a biometric unit (antibody) with a signal amplification element (enzyme) through the biomineralization process. Specifically, the critical factors affecting antibody recognition activity in the formation mechanism of AAHNFs are investigated. The designed AAHNFs retain antibody recognition ability with enhanced protection for encapsulated proteins against high temperature, organic solvents, and long-term storage, facilitating the selective construction of lock structures against antigens. Additionally, a colorimetric immunosensor based on AAHNFs was developed. After ascorbic acid 2-phosphate hydrolysis by alkaline phosphatase (ALP), the generated ascorbic acid decomposes I2 to I-, inducing the localized surface plasmon resonance in the silver nanoplate, which is effectively tuned through shape conversion to develop the sensor. Further, a 3D-printed portable device is fabricated, integrated with a smartphone sensing platform, and applied to the data of collection and analysis. Notably, the immunosensor exhibits improved analytical performance with a 0.1-6.25 ng·mL-1 detection range and a 0.06 ng·mL-1 detection limit for quantitative saxitoxin (STX) analysis. The average recoveries of STX in real samples ranged from 85.9% to 105.9%. This study presents a more in-depth investigation of the recognition element performance, providing insights for improved antibody performance in practical applications.

PfAgo-based dual signal amplification biosensor for rapid and highly sensitive detection of alkaline phosphatase activity.

A Pyrococcus furiosus Argonaute (PfAgo)-based biosensor is presented for alkaline phosphatase (ALP) activity detection in which the ALP-catalyzed hydrolysis of 3'-phosphate-modified functional DNA activates the strand displacement amplification, and the amplicon mediates the fluorescent reporter cleavage as a guide sequence of PfAgo. Under the dual amplification mode of PfAgo-catalyzed multiple-turnover cleavage activity and pre-amplification technology, the developed method was successfully applied to ALP activity determination with a detection limit (LOD) of 0.0013 U L-1 (3σ) and a detection range of 0.0025 to 1 U L-1 within 90 min. The PfAgo-based method exhibits satisfactory analytic performance in the presence of potential interferents and in complex human serum samples. The proposed method shows several advantages, such as rapid analysis, high sensitivity, low-cost, and easy operation, and has great potential in disease evolution fundamental studies and clinical diagnosis applications.

Hydroxyl radical-mediated synthesis of carbonyl functionalized graphene quantum dots-like as enzyme mimics with tunable fluorescence emission.

The synthesis of graphene quantum dots-like enriched with specific oxygenated groups (o-GQDs) exhibiting great catalytic performance offers a promising tool for diagnosis and biomedicine, but introducing specific oxygen groups remains a challenge. Here, we propose a mild synthetic protocol for producing regulated fluorescence emission (from blue to yellow) carbonyl functionalized GQDs with double catalytic function through Fe3O4-catalyzed hydroxyl radical (·OH) oxidation the precursors like graphene oxide, polyaniline (PANI) and polydopamine (PDA). The method can be carried out at room temperature than the traditional high-temperature oxidation in concentrated acid. Interestingly, o-GQDs exhibit excellent peroxidase (POD)- and ascorbate oxidase-like activity. XPS characterization showed a significant increase in carbonyl content in o-GQDs compared to the precursor, even a 14-fold increase in blue-emitting iron-doped GQDs (b-Fe-GQDs). The introduction of Fe3O4 during the synthesis process results in a minor degree of Fe doping, which enhances the catalytic activity of b-Fe-GQDs through coordination with N. Based on this feature, highly sensitive single-signal and ultra-selective dual-signal methods for alkaline phosphatase detection were developed. This low cost and safe synthesis strategy paves the way for practical usage of o-GQDs.

Closed Bipolar Nanoelectrode Array for Ultra-Sensitive Detection of Alkaline Phosphatase and Two-Dimensional Imaging of Epidermal Growth Factor Receptors.

The combination of closed bipolar electrodes (cBPE) with electrochemiluminescence (ECL) imaging has demonstrated remarkable capabilities in the field of bioanalysis. Here, we established a cBPE-ECL platform for ultrasensitive detection of alkaline phosphatase (ALP) and two-dimensional imaging of epidermal growth factor receptor (EGFR). This cBPE-ECL system consists of a high-density gold nanowire array in anodic aluminum oxide (AAO) membrane as the cBPE coupled with ECL of highly luminescent cadmium selenide quantum dots (CdSe QDs) luminophores to achieve cathodic electro-optical conversion. When an enzyme-catalyzed amplification effect of ALP with 4-aminophenyl phosphate monosodium salt hydrate (p-APP) as the substrate and 4-aminophenol (p-AP) as the electroactive probe is introduced, a significant improvement of sensing sensitivity with a detection limit as low as 0.5 fM for ALP on the cBPE-ECL platform can be obtained. In addition, the cBPE-ECL sensing system can also be used to detect cancer cells with an impressive detection limit of 50 cells/mL by labeling ALP onto the EGFR protein on A431 human epidermal cancer cell membranes. Thus, two-dimensional (2D) imaging of the EGFR proteins on the cell surface can be achieved, demonstrating that the established cBPE-ECL sensing system is of high resolution for spatiotemporal cell imaging.

Construction of the fluorescence sensing platform with a bifunctional Cu@MOF nanozyme for determination of alkaline phosphatase and its inhibitor.

In this work, a novel and sensitive fluorescence sensing system for alkaline phosphatase (ALP) was constructed using a bifunctional copper metal-organic framework (Cu@MOF) nanozyme, which had excellent oxidase-mimetic activity and fluorescence properties. Owing to the presence of 2-amino-1,4-benzenedicarboxylic acid (1,4-BDC-NH2) ligand, Cu@MOF displays excellent fluorescence performance at 444 nm. Additionally, Cu2+ endows the oxidase-like activity of Cu@MOF, which could trigger p-phenylenediamine (PPD) to be oxidized to a brown product (PPDox) and quench the photoluminescence of Cu@MOF through the inner filtration effect (IFE). As the preferential affinity of ATP for Cu2+, the catalytic activity of Cu@MOF was significantly reduced once ATP was added, thus PPD could not be oxidized and fluorescence was recovered. In the presence of ALP, ATP was hydrolyzed to adenosine and Pi, which allowed Cu@MOF to regain its catalytic activity and continued to catalyze the generation of PPDox. The fluorescence of Cu@MOF was therefore weakened once again. The ALP activity was directly proportional to the degree of decrease in fluorescence intensity. Thus, this novel fluorescence sensing strategy had a linear range of 0.5-60 U/L and the limit of detection was 0.14 U/L. The established sensing method could also be used to for ALP inhibitors screening, and achieved satisfactory results in determining the level of ALP activity in human serum.

Multiquenching-Based Aggregation-Induced Electrochemiluminescence Sensing for Highly Sensitive Detection of the SARS-CoV-2 N Protein.

The rapid epidemic around the world of coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, proves the need and stimulates efforts to explore efficient diagnostic tests for the sensitive detection of the SARS-CoV-2 virus. An aggregation-induced electrochemiluminescence (AIECL) sensor was developed for the ultrasensitive detection of the SARS-CoV-2 nucleocapsid (N) protein in this work. Tetraphenylethylene doped in zeolite imidazole backbone-90 (TPE-ZIF-90) showed highly efficient aggregation-induced emission (AIE) to endow TPE-ZIF-90 with high ECL intensity. Upon the capture of the SARS-CoV-2 N protein by immune recognition, an alkaline phosphatase (ALP)-modified gold nanoparticle (AuNP)-decorated zinc oxide (ZnO) nanoflower (ALP/Au-ZnO) composite was introduced on the sensing platform, which catalyzed L-ascorbate-2-phosphate trisodium salt (AA2P) to produce PO43- and ascorbic acid (AA). Based on a multiquenching of the ECL signal strategy, including resonance energy transfer (RET) between TPE-ZIF-90 and Au-ZnO, disassembly of TPE-ZIF-90 triggered by the strong coordination between PO43- and Zn2+, and RET between TPE-ZIF-90 and AuNPs produced in situ by the AA reductive reaction, the constructed AIECL sensor achieved highly sensitive detection of the SARS-CoV-2 N protein with a low limit of detection of 0.52 fg/mL. With the merits of high specificity, good stability, and proven application ability, the present RET- and enzyme-triggered multiquenching AIECL sensor may become a powerful tool in the field of SARS-CoV-2 virus diagnosis.

A ratiometric fluorescence sensor for detection of organophosphorus pesticides based on enzyme-regulated multifunctional Fe-based metal-organic framework.

The residues of organophosphorus pesticides (OPs) are increasing environmental pollution and public health concerns. Thus, the development of simple, convenient and sensitive method for detection of OPs is crucial. Herein, a multifunctional Fe-based MOF with fluorescence, catalytic and adsorption, is synthesized by a simple one-pot hydrothermal method. The ratiometric fluorescence sensor for detection of OPs is constructed by using only one multifunctional sensing material. The NH2-MIL-101(Fe) is able catalyze the o-phenylenediamine (OPD) into 2,3-diaminophenazine (DAP) in the presence of H2O2. The generated DAP can significantly quench the intrinsic fluorescence of NH2-MIL-101(Fe) by the fluorescence resonance energy transfer (FRET) and internal filtration effect (IFE), while producing a new measurable fluorescence. Without immobilization or molecular imprinting, pyrophosphate ion (PPi) can inhibit the peroxidase-like activity of the NH2-MIL-101(Fe) by chelating with Fe3+/Fe2+ redox couple. Moreover, PPi can also be hydrolyzed by alkaline phosphatase (ALP), the presence of OPs inhibits the activity of ALP, resulting in the increase of extra PPi preservation and signal changes of ratiometric fluorescence, the interactions of ALP with different OPs are explored by molecular docking, the OPs (e.g., glyphosate) interact with crucial amino acid residues (Asp, Ser, Ala, Lys and Arg) are indicated. The proposed sensor exhibits excellent detection performance for OPs with the detection limit of 18.7 nM, which provides a promising strategy for detection of OPs.

A Programmable Microfluidic Paper-Based Analytical Device for Simultaneous Colorimetric and Photothermal Visual Sensing of Multiple Enzyme Activities.

New strategies for the simultaneous and portable detection of multiple enzyme activities are highly desirable for clinical diagnosis and home care. However, the methods developed thus far generally suffer from high costs, cumbersome procedures, and heavy reliance on large-scale instruments. To satisfy the actual requirements of rapid, accurate, and on-site detection of multiple enzyme activities, we report herein a smartphone-assisted programmable microfluidic paper-based analytical device (µPAD) that utilizes colorimetric and photothermal signals for simultaneous, accurate, and visual quantitative detection of alkaline phosphatase (ALP) and butyrylcholinesterase (BChE). Specifically, the operation of this µPAD sensing platform is based on two sequential steps. Cobalt-doped mesoporous cerium oxide (Co-m-CeO2) with remarkable peroxidase-like activities under neutral conditions first catalytically decomposes H2O2 for effectively converting colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB). The subsequent addition of ALP or BChE to their respective substrates produces a reducing substance that can somewhat inhibit the oxTMB transformation for compromised colorimetric and photothermal signals of oxTMB. Notably, these two-step bioenzyme-nanozyme cascade reactions strongly support the straightforward and excellent processability of this platform, which exhibit lower detection limits for ALP and BChE with a detection limit for BChE an order of magnitude lower than those of the other reported paper-based detection methods. The practicability and efficiency of this platform are further demonstrated through the analysis of clinical serum samples. This innovative platform exhibits great potential as a facile yet robust approach for simultaneous, accurate, and on-site visual detection of multiple enzyme activities in authentic samples.

Catalyzing precision: unraveling the diagnostic conundrum of tunisian familial hypophosphatasia case through integrative clinical and molecular approaches.

Familial Hypophosphatasia presents a complex diagnostic challenge due to its wide-ranging clinical manifestations and genetic heterogeneity. This study aims to elucidate the molecular underpinnings of familial Hypophosphatasia within a Tunisian family harboring a rare c.896 T > C mutation in the ALPL gene, offering insights into genotype-phenotype correlations and potential therapeutic avenues. The study employs a comprehensive approach, integrating biochemical examination, genetic analysis, structural modeling, and functional insights to unravel the impact of this rare mutation. Genetic investigation revealed the presence of the p.Leu299Pro mutation within the ALPL gene in affected family members. This mutation is strategically positioned in proximity to both the catalytic site and the metal-binding domain, suggesting potential functional consequences. Homology modeling techniques were employed to predict the 3D structure of TNSALP, providing insights into the structural context of the mutation. Our findings suggest that the mutation may induce conformational changes in the vicinity of the catalytic site and metal-binding domain, potentially affecting substrate recognition and catalytic efficiency. Molecular dynamics simulations were instrumental in elucidating the dynamic behavior of the tissue-nonspecific alkaline phosphatase isozyme (TNSALP) in the presence of the p.Leu299Pro mutation. The simulations indicated alterations in structural flexibility near the mutation site, with potential ramifications for the enzyme's overall stability and function. These dynamic changes may influence the catalytic efficiency of TNSALP, shedding light on the molecular underpinnings of the observed clinical manifestations within the Tunisian family. The clinical presentation of affected individuals highlighted significant phenotypic heterogeneity, underscoring the complex genotype-phenotype correlations in familial Hypophosphatasia. Variability in age of onset, severity of symptoms, and radiographic features was observed, emphasizing the need for a nuanced understanding of the clinical spectrum associated with the p.Leu299Pro mutation. This study advances our understanding of familial Hypophosphatasia by delineating the molecular consequences of the p.Leu299Pro mutation in the ALPL gene. By integrating genetic, structural, and clinical analyses, we provide insights into disease pathogenesis and lay the groundwork for personalized therapeutic strategies tailored to specific genetic profiles. Our findings underscore the importance of comprehensive genetic and clinical evaluation in guiding precision medicine approaches for familial Hypophosphatasia.

Enzyme-Activated Biomimetic Vesicles Confining Mineralization for Bone Maturation.

Inspired by the crucial role of matrix vesicles (MVs), a series of biomimetic vesicles (BVs) fabricated by calcium glycerophosphate (CaGP) modified polyurethane were designed to mediate the mineralization through in situ enzyme activation for bone therapy. In this study, alkaline phosphatase (ALP) was harbored in the porous BVs by adsorption (Ad-BVs) or entrapment (En-BVs). High encapsulation of ALP on En-BVs was effectively self-activating by calcium ions of CaGP-modified PU that specifically hydrolyzed the organophosphorus (CaGP) to inorganic phosphate, thus promoting the formation of the highly oriented bone-like apatite in vitro. Enzyme-catalyzed kinetics confirms the regulation of apatite crystallization by the synergistic action of self-activated ALP and the confined microcompartments of BVs. This leads to a supersaturated microenvironment, with the En-BVs group exhibiting inorganic phosphate (Pi) levels 4.19 times higher and Ca2+ levels 3.67 times higher than those of simulated body fluid (SBF). Of note, the En-BVs group exhibited excellent osteo-inducing differentiation of BMSCs in vitro and the highest maturity with reduced bone loss in rat femoral defect in vivo. This innovative strategy of biomimetic vesicles is expected to provide valuable insights into the enzyme-activated field of bone therapy.

Automated System for Attomolar-Level Detection of MiRNA as a Biomarker for Influenza A Virus.

We have developed an automated sensing system for the repeated detection of a specific microRNA (miRNA) of the influenza A (H1N1) virus. In this work, magnetic particles functionalized with DNAs, target miRNAs, and alkaline phosphate (ALP) enzymes formed sandwich structures. These particles were trapped on nickel (Ni) patterns of our sensor chip by an external magnetic field. Then, additional electrical signals from electrochemical markers generated by ALP enzymes were measured using the sensor, enabling the highly sensitive detection of target miRNA. The magnetic particles used on the sensor were easily removed by applying the opposite direction of external magnetic fields, which allowed us to repeat sensing measurements. As a proof of concept, we demonstrated the detection of miRNA-1254, one of the biomarkers for the H1N1 virus, with a high sensitivity down to 1 aM in real time. Moreover, our sensor could selectively detect the target from other miRNA samples. Importantly, our sensor chip showed reliable electrical signals even after six repeated miRNA sensing measurements. Furthermore, we achieved technical advances to utilize our sensor platform as part of an automated sensing system. In this regard, our reusable sensing platform could be utilized for versatile applications in the field of miRNA detection and basic research.

Fucosylation and galactosylation in N-glycans of bovine intestinal alkaline phosphatase and their role in its enzymatic activity.

Bovine intestinal alkaline phosphatase (biALP), a membrane-bound plasma metalloenzyme, maintains intestinal homeostasis, regulates duodenal surface pH, and protects against infections caused by pathogenic bacteria. The N-glycans of biALP regulate its enzymatic activity, protein folding, and thermostability, but their structures are not fully reported. In this study, the structures and quantities of the N-glycans of biALP were analyzed by liquid chromatography-electrospray ionization-high energy collision dissociation-tandem mass spectrometry. In total, 48 N-glycans were identified and quantified, comprising high-mannose [6 N-glycans, 33.1 % (sum of relative quantities of each N-glycan)], hybrid (6, 11.9 %), and complex (36, 55.0 %) structures [bi- (13, 26.1 %), tri- (16, 21.5 %), and tetra-antennary (7, 7.4 %)]. These included bisecting N-acetylglucosamine (33, 56.6 %), mono-to tri-fucosylation (32, 53.3 %), mono-to tri-α-galactosylation (16, 20.7 %), and mono-to tetra-ß-galactosylation (36, 58.5 %). No sialylation was identified. N-glycans with non-bisecting GlcNAc (9, 10.3 %), non-fucosylation (10, 13.6 %), non-α-galactosylation (26, 46.2 %), and non-ß-galactosylation (6, 8.4 %) were also identified. The activity (100 %) of biALP was reduced to 37.3 ± 0.2 % (by de-fucosylation), 32.7 ± 2.9 % (by de-α-galactosylation), and 0.2 ± 0.2 % (by de-ß-galactosylation), comparable to inhibition by 10-4 to 101 mM EDTA, a biALP inhibitor. These results indicate that fucosylated and galactosylated N-glycans, especially ß-galactosylation, affected the activity of biALP. This study is the first to identify 48 diverse N-glycan structures and quantities of bovine as well as human intestinal ALP and to demonstrate the importance of the role of fucosylation and galactosylation for maintaining the activity of biALP.

Atomically Co-dispersed nitrogen-doped carbon for sensitive electrochemical immunoassay of breast cancer biomarker CA15-3.

The development of an ultrasensitive and precise measurement of a breast cancer biomarker (cancer antigen 15-3; CA15-3) in complex human serum is essential for the early diagnosis of cancer in groups of healthy populations and the treatment of patients. However, currently available testing technologies suffer from insufficient sensitivity toward CA15-3, which severely limits early large-scale screening of breast cancer patients. We report a versatile electrochemical immunoassay method based on atomically cobalt-dispersed nitrogen-doped carbon (Co-NC)-modified disposable screen-printed carbon electrode (SPCE) with alkaline phosphatase (ALP) and its metabolite, ascorbic acid 2-phosphate (AAP), as the electrochemical labeling and redox signaling unit for sensitive detection of low-abundance CA15-3. During electrochemical detection by differential pulse voltammetry (DPV), it was found that the Co-NC-SPCE electrode did not have a current signal response to the AAP substrate; however, it had an extremely favorable response current to ascorbic acid (AA). Based on the above principle, the target CA15-3-triggered immunoassay enriched ALP-catalyzed AAP produces a large amount of AA, resulting in a significant change in the system current signal, thereby realizing the highly sensitive detection of CA15-3. Under the optimal AAP substrate concentration and ALP catalysis time, the Co-NC-SPCE-based electrochemical immunoassay demonstrated a good DPV current for CA15-3 in the assay interval of 1.0 mU/mL to 10,000 mU/mL, with a calculated limit of detection of 0.38 mU/mL. Since Co-NC-SPCE has an excellent DPV current response to AA and employs split-type scheme, the constructed electrochemical immunoassay has the merits of high preciseness and anti-interference, and its clinical diagnostic results are comparable to those of commercial kits.

Based ATP-gating mechanism for detection of alkaline phosphatase in single-glass micropipettes functionalized by three-dimensional DNA network.

The construction of gating system in artificial channels is a cutting-edge research direction in understanding biological process and application sensing. Here, by mimicking the gating system, we report a device that easily synthesized single-glass micropipettes functionalized by three-dimensional (3D) DNA network, which triggers the gating mechanism for the detection of biomolecules. Based on this strategy, the gating mechanism shows that single-glass micropipette assembled 3D DNA network is in the "OFF" state, and after collapsing in the presence of ATP, they are in the "ON" state, at which point they exhibit asymmetric response times. In the "ON" process of the gating mechanism, the ascorbic acid phosphate (AAP) can be encapsulated by a 3D DNA network and released in the presence of adenosine triphosphate (ATP), which initiates a catalyzed cascade reaction under the influence of alkaline phosphatase (ALP). Ultimately, the detection of ALP can be responded to form the fluorescence signal generated by terephthalic acid that has captured hydroxyl radicals, which has a detection range of 0-250 mU/mL and a limit of detection of 50 mU/mL. This work provides a brand-new way and application direction for research of gating mechanism.

Conformation-Switchable Polypeptides as Molecular Gates for Controllable Drug Release.

The control over secondary structure has been widely studied to regulate the properties of polypeptide materials, which is used to change their functions in situ for various biomedical applications. Herein, we designed and constructed enzyme-responsive polypeptides as gating materials for mesoporous silica nanoparticles (MSNs), which underwent a distorted structure-to-helix transition to promote the release of encapsulated drugs. The polypeptide conjugated on the MSN surface adopted a negatively charged, distorted, flexible conformation, covering the pores of MSN to prevent drug leakage. Upon triggering by alkaline phosphatase (ALP) overproduced by tumor cells, the polypeptide transformed into positively charged, α-helical, rigid conformation with potent membrane-penetrating capabilities, which protruded from the MSN surface to uncover the pores. Such a transition thus enabled cancer-selective drug release and cellular internalization to efficiently kill tumor cells. This study highlights the important role of chain flexibility in modulating the biological function of polypeptides and provides a new application paradigm for synthetic polypeptides with secondary-structure transition.

Detection of alkaline phosphatase activity with a CsPbBr3/Y6 heterojunction-based photoelectrochemical sensor.

An ultra-sensitive photoelectrochemical (PEC) sensor based on perovskite composite was developed for the determination of alkaline phosphatase (ALP) in human serum. In contrast to CsPbBr3 or Y6 that generated anodic current, the heterojunction of CsPbBr3/Y6 promoted photocarriers to separate and generated cathodic photocurrent. Ascorbic acid (AA) was produced by ALP hydrolyzing L-ascorbic acid 2-phosphate trisodium salt (AAP), which can combine with the holes on the photoelectrode surface, accelerating the transmission of photogenerated carriers, leading to enhanced photocurrent intensity. Thus, the enhancement of PEC current was linked to ALP activity. The PEC sensor exhibits good sensitivity for detection of ALP owing to the unique photoelectric properties of the CsPbBr3/Y6 heterojunction. The detection limit of the sensor was 0.012 U·L-1 with a linear dynamic range of 0.02-2000 U·L-1. Therefore, this PEC sensing platform shows great potential for the development of different PEC sensors.