DNA-directed immobilization fluorescent immunoarray for multiplexed antibiotic residue determination in milk.

The presence of antibiotic residues in cow's milk entails high risk for consumers, the dairy industry, and the environment. Therefore, the development of highly specific and sensitive screening tools for the rapid and cost-effective identification of traces of these compounds is urgently needed. A multiplexed screening platform utilizing DNA-directed immobilization (DDI) was developed aiming to detect three classes of antibiotic residues (fluoroquinolones, sulfonamides, and tylosin) prevalently found in milk. Throughout this work, each oligonucleotide sequence was conjugated to a different hapten molecule, while the three complementary strands were immobilized in 24 independent microarray chips on a single glass slide. First, the array was incubated with the pool of hapten-oligonucleotide conjugate site encoded the signal through DNA hybridization. Next, commercial milk samples were incubated with the cocktail of monoclonal antibodies following a secondary fluorophore-labeled antibody which was required for fluorescent readout. Direct sample detection was achieved in milk diluting 20 times in assay buffer. The limits of detection (LODs) reached were 1.43 µg kg-1, 1.67 µg kg-1, and 0.89 µg kg-1 for TYLA, STZ, and CIP, respectively, which represented in raw milk 7.15 µg kg-1, 8.35 µg kg-1, and 4.45 µg kg-1 for TYLA, STZ, and CIP, respectively, that are below the EU regulatory limits. Cross-reactivity profiles were evaluated against the family of structurally related antibiotics in order to demonstrate the capability to detect antibiotics from the same family of compounds. A pre-validation study was performed by spiking 20 blind samples above and below the maximum residue limits established by the EU guidelines. The system was successfully implemented towards randomized sample classification as compliant or non-compliant. The proposed DDI-based immunoarray provides a fast and cost-effective alternative to obtain semi-quantitative information about the presence of three veterinary residues simultaneously in milk samples.

Controllable and high-performance immobilized enzyme reactor: DNA-directed immobilization of multienzyme in polyamidoamine dendrimer-functionalized capillaries.

In recent years, CE-integrated immobilized enzyme reactors (IMERs) for single-enzyme immobilization have attracted considerable attention. However, there has been little research on multienzyme immobilization in CE. Here, we introduce a method for fabricating a CE-integrated IMER, using DNA-directed immobilization to fix glucose oxidase and horseradish peroxidase in the capillary, which had been functionalized with polyamidoamine dendrimer (PAMAM). Owing to the reversibility of DNA hybridization, the reactor is capable of dynamic immobilization. Moreover, by introducing the PAMAM, the loading capacity of the IMER is greatly enhanced, and the PAMAM can spontaneously form complexes with DNA and then contribute to the efficiency and stability of the reactor. After 25 days storage, the prepared IMER ultimately retained approximately 70% of its initial activity. We also used the IMER to detect glucose, and the favorable linearity was obtained over the concentration range of 0.78-12.5 mM, with an LOD of 0.39 mM, demonstrating that the CE-integrated IMER can be applied to actual samples. We believe that this strategy can be extended to other multienzyme immobilization systems, and CE-integrated IMERs are potentially useful in a wide range of biochemical research applications.

Photo-Induced Click Chemistry for DNA Surface Structuring by Direct Laser Writing.

Oligonucleotides containing photo-caged dienes were prepared and shown to react quantitatively in a light-induced Diels-Alder cycloaddition with functional maleimides in aqueous solution within minutes. Due to its high yield and fast rate, the reaction was exploited for DNA surface patterning with sub-micrometer resolution employing direct laser writing (DLW). Functional DNA arrays were written by direct laser writing (DLW) in variable patterns, which were further encoded with fluorophores and proteins through DNA directed immobilization. This mild and efficient light-driven platform technology holds promise for the fabrication of complex bioarrays with sub-micron resolution.

A DNA-based nano-immunoassay for the label-free detection of glial fibrillary acidic protein in multicell lysates.

We have developed a quantitative approach to eventually enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells. Through DNA-directed immobilization of DNA-protein conjugates we immobilized antibodies specific for a certain protein of interest, on a complementary DNA nanoarray fabricated by means of nanografting, a nanolithography technique based on atomic force microscopy (AFM). The proof of concept was realized for glial fibrillary acidic protein (GFAP), a biomarker crucial in cell's differentiation of astrocytes, and functional to grade classification of gliomas, the most common of primary malignant brain tumors. The efficiency of the nano-immuno sensing was tested by obtaining the immobilization of purified recombinant GFAP protein at different concentration in a standard solution then in a cellular lysate. A comparison of sensitivity between our technique and conventional ELISA assays is provided at the end of the paper. FROM THE CLINICAL EDITOR: This team developed a quantitative approach to enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells, demonstrating the utility of this DNA-based nano-immunoassay in the detection of GFAP.

DNA flexible chain modified MOFs as a versatile platform for chemoenzymatic cascade reactions in glucose catalysis.

Glucose oxidase (GOD) is widely used in the pharmaceutical industry, fermentation products and glucose biosensors for its essential role in catalyzing the conversion of glucose to gluconic acid and hydrogen peroxide (H2O2). As H2O2 is the by-product and will have a toxic effect on glucose oxidase, so introducing another enzyme that could consume H2O2 to form an enzymatic cascade reaction is a practical solution. However, this decision will lead to extra expenses and complex condition optimization such as the specific mass ratio, temperature and pH to improve the activity, stability and recyclability. Herein, we describe a mild and versatile strategy by anchoring GOD on carboxyl-activated MOF (Cu-TCPP(Fe)) through DNA-directed immobilization (DDI) technology. Robust MOF nanosheets were utilized as not only the carrier for the immobilization of GOD, but also a peroxidase-like catalyst for the decomposition of H2O2 to reduce its harmful impacts. In this work, the immobilized GOD retained 55.78% of its initial activity after being used for 7 times. More than 60% of the immobilized enzyme's catalytic activity was still maintained after 96 h of being stored at 50 â„ƒ. This study provides a new idea for preparing immobilized enzymes with enhanced stability, fast diffusion and high activity, which can be used in fields such as biocatalysis and biotechnology.

A Careful Insight into DDI-Type Receptor Layers on the Way to Improvement of Click-Biology-Based Immunosensors.

Protein-based microarrays are important tools for high-throughput medical diagnostics, offering versatile platforms for multiplex immunodetection. However, challenges arise in protein microarrays due to the heterogeneous nature of proteins and, thus, differences in their immobilization conditions. This article advocates DNA-directed immobilization (DDI) as a solution, emphasizing its rapid and cost-effective fabrication of biosensing platforms. Thiolated single-stranded DNA and its analogues, such as ZNA® and PNA probes, were used to immobilize model proteins (anti-CRP antibodies and SARS-CoV nucleoprotein). The study explores factors influencing DDI-based immunosensor performance, including the purity of protein-DNA conjugates and the stability of their duplexes with DNA and analogues. It also provides insight into backfilling agent type and probe surface density. The research reveals that single-component monolayers lack protection against protein adsorption, while mixing the probes with long-chain ligands may hinder DNA-protein conjugate anchoring. Conventional DNA probes offer slightly higher surface density, while ZNA® probes exhibit better binding efficiency. Despite no enhanced stability in different ionic strength media, the cost-effectiveness of DNA probes led to their preference. The findings contribute to advancing microarray technology, paving the way for new generations of DDI-based multiplex platforms for rapid and robust diagnostics.

Construction of co-immobilized multienzyme systems using DNA-directed immobilization technology and multifunctionalized nanoparticles.

The multienzyme co-immobilization systems with high cascade catalytic efficiency and selectivity have attracted considerable attention. In this study, through DNA-directed immobilization (DDI) technology, two model enzymes, glucose oxidase (GOD) and horseradish peroxide (HRP) were co-immobilized on the multifunctional silica nanoparticles (DDI enzyme). In addition to the directional distribution promoted by DNA complementary chains, the multienzyme system allowed the control of the stoichiometric ratio of the enzymes by adjusting the ratio of amino/carboxyl groups. The optimal mole ratio of GOD/HRP was 1:2, while the protein loading amount could reach 8.06 mg·g-1. Compared with the conventional direct adsorption, the catalytic activity of the DDI enzyme was 2.49 times higher. Moreover, with the enhancement of thermal and mechanical stability, the DDI enzyme could still retain at least 50% of its initial activity after 12 cycles. Accompanied by an excellent response and good selectivity, the constructed multienzyme systems simultaneously showed the potential as a glucose detector. Therefore, based on the DDI technology, the highly efficient multienzyme co-immobilization system could be further extended for a wider range of research fields.

DNA-Directed Protein Anchoring on Oligo/Alkanethiol-Coated Gold Nanoparticles: A Versatile Platform for Biosensing Applications.

Herein, we report on a smart biosensing platform that exploits gold nanoparticles (AuNPs) functionalized through ssDNA self-assembled monolayers (SAM) and the DNA-directed immobilization (DDI) of DNA-protein conjugates; a novel, high-sensitivity optical characterization technique based on a miniaturized gel electrophoresis chip integrated with online thermal lens spectrometry (MGEC-TLS), for the high-sensitivity detection of antigen binding events. Specifically, we characterized the physicochemical properties of 20 nm AuNPs covered with mixed SAMs of thiolated single-stranded DNA and bio-repellent molecules, referred to as top-terminated oligo-ethylene glycol (TOEG6), demonstrating high colloidal stability, optimal binder surface density, and proper hybridization capacity. Further, to explore the design in the frame of cancer-associated antigen detection, complementary ssDNA fragments conjugated with a nanobody, called C8, were loaded on the particles and employed to detect the presence of the HER2-ECD antigen in liquid. At variance with conventional surface plasmon resonance detection, MGEC-TLS characterization confirmed the capability of the assay to titrate the HER2-ECD antigen down to concentrations of 440 ng/mL. The high versatility of the directed protein-DNA conjugates immobilization through DNA hybridization on plasmonic scaffolds and coupled with the high sensitivity of the MGEC-TLS detection qualifies the proposed assay as a potential, easily operated biosensing strategy for the fast and label-free detection of disease-relevant antigens.

DNA-directed coimmobilization of multiple enzymes on organic-inorganic hybrid DNA flowers.

The artificial multienzyme systems developed by mimicking nature has attracted much interest. However, precisely controlled compositions and ratios of multienzymatic co-immobilization systems are still limited by the indistinguishable nature of enzymes. Herein, a strategy for fabricating DNA-directed immobilization of horseradish peroxidase (HRP) and glucose oxidase (GOx) on hybrid DNA nanoflowers (GOx-HRP@hDFs) is presented. The preparation of micron-sized hybrid DNA flowers (hDFs) begins with the predetermined repeatable polymer-like DNA sequences which contained two strands. The hDFs structure is generated through one-pot rolling circle amplification (RCA) and self-assembly with magnesium pyrophosphate inorganic crystals. Based on the rigid-base pairing, GOx and HRP conjugated with sequences complementary to strands would be anchored to the predesigned locations, respectively. By adjusting the loading amount/ratio of enzymes properly, the maximal catalytic efficiency can be precisely regulated. The reaction activity of GOx-HRP@hDFs was 7.4 times higher than that of the free GOx-HRP under the optimal mole ratio (GOx/HRP 4:1). In addition, this multienzyme catalyst system exhibits excellent precision, specificity, reproducibility, and long-term storage stability when applied to real human blood samples. The preceding results validate that GOx-HRP@hDFs are promising candidates for personal diabetes detection.

Advantageous antibody microarray fabrication through DNA-directed immobilization: A step toward use of extracellular vesicles in diagnostics.

Microarrays were introduced to run multiple assays on a single platform. Since then, researchers developed DNA and protein microarrays to study both transcription and expression of genes. Protein microarray technology represents a powerful tool to get an insight into living systems. However, despite their enormous potential, the fabrication of protein arrays is affected by technological hurdles that limit their application. One of the significant challenges is the immobilization of proteins on solid surfaces. To overcome this limitation, DNA-directed immobilization (DDI) of proteins, an approach that exploits DNA-protein conjugates to transform DNA microarrays into a protein array, has been developed. The adoption of DDI is limited, as this approach requires the synthesis of DNA-protein conjugates. Herein, we introduce an optimized general protocol for DNA-protein ligation, and demonstrate the use of conjugates to convert DNA arrays into antibody microarrays. Arrays obtained through DDI were used to capture and characterize extracellular vesicles (EVs), an emerging class of biomarkers. The proposed platform was tested against commercially available antibody microarrays, showing good performance combined with ease of fabrication.

[Advances in capillary-based immobilized enzyme microreactor based on DNA-directed immobilization].

Life processes such as metabolism and energy conversion are catalyzed by biological enzymes. The changes of enzymatic activity in organisms can lead various diseases. Thus, it is imperative to develop novel methods of analyzing enzymatic activities for gaining deeper insights into metabolic processes, disease diagnosis, and drug development. Capillary electrophoresis (CE) has the advantages of high separation efficiency, fast analysis speed, and simple operation; moreover, it requires less sample and can be combined with a variety of detection methods. Therefore, CE has attracted increasing attention for enzyme analysis. Enzyme analysis based on CE mainly includes off-line mode and on-line mode. In the off-line mode, the enzyme and substrate are incubated outside the capillary, and then the product is introduced into the CE for analysis. In the on-line mode, the capillary is not only used as a separation channel, but also as an enzyme reaction site. Therefore, the on-line mode facilitates all steps of enzymatic hydrolysis, separation, and detection within a capillary. In the on-line mode, homogeneous analysis method, electrophoretically mediated microanalysis (EMMA), and heterogeneous analysis method, immobilized enzyme microreactor (IMER), were developed. The on-line enzyme analysis method of IMER combined with capillary electrophoresis (CE-IMER) was developed into a mainstream enzyme analysis method. CE-IMER combines the advantages of immobilized enzyme and CE. By immobilizing the free enzyme in capillary, it can not only significantly improve the stability and reusability of enzyme, but also enables the automatic enzyme analysis at nanoscale. This can significantly reduce the cost of enzyme analysis. Although, there are numerous methods to prepare new IMER for enzyme analysis by CE, preparing CE-IMER with good performance, reusability, large enzyme loading, and high degree of automation is the focus of research in this field. DNA-directed immobilization (DDI) makes use of the complementary base pairs (A-T, C-G) of DNA molecules to specifically immobilize biomacromolecules under mild physiological conditions. The enzyme can be immobilized on the carrier surface by DDI and the short double helix DNA molecules possess strong mechanical strength and physicochemical stability. This can form an enzyme microarray, reduce the resistance of mass transfer, improve the contact between enzyme and substrate, and promote the enzymatic analysis process. Compared with the traditional immobilization methods of adsorption, crosslinking, encapsulation, and covalent bonding, DDI can be operated under mild physiological conditions. Further, this can significantly reduce the influence of the immobilization process on the activity, conformation, and stability of the enzyme. Meanwhile, the reversible immobilization process of DDI can regenerate the surface of the carrier, thereby significantly reducing the economic and time cost of IMER preparation. Therefore, DDI is an ideal method to prepare IMER. In this article, the preliminary research and progress of our research group in the field of IMER preparation by DDI technology are presented. At present, the research on the preparation of novel IMER based on DNA nanotechnology, such as DDI, is in the initial stage and there is much scope for development and research. Based on the previous studies, we can focus on the following aspects: (1) building a more efficient catalytic IMER cascade reaction system by immobilizing target enzymes in specific regions of the capillary based on DDI; (2) aiming at the problems existing in the preparation of IMER, such as stability, enzymatic activity, and enzyme immobilization capacity, while taking advantages of DNA structure and nanomaterials to prepare novel IMERs to promote the wide application of CE-IMER in enzyme analysis.

From DNA Nanotechnology to Material Systems Engineering.

In the past 35 years, DNA nanotechnology has grown to a highly innovative and vibrant field of research at the interface of chemistry, materials science, biotechnology, and nanotechnology. Herein, a short summary of the state of research in various subdisciplines of DNA nanotechnology, ranging from pure "structural DNA nanotechnology" over protein-DNA assemblies, nanoparticle-based DNA materials, and DNA polymers to DNA surface technology is given. The survey shows that these subdisciplines are growing ever closer together and suggests that this integration is essential in order to initiate the next phase of development. With the increasing implementation of machine-based approaches in microfluidics, robotics, and data-driven science, DNA-material systems will emerge that could be suitable for applications in sensor technology, photonics, as interfaces between technical systems and living organisms, or for biomimetic fabrication processes.

DNA-directed trypsin immobilization on a polyamidoamine dendrimer-modified capillary to form a renewable immobilized enzyme microreactor.

A novel type of trypsin capillary microreactor was developed based on a DNA-directed immobilization (DDI) technique applied to a fused-silica capillary modified with polyamidoamine (PAMAM) dendrimers. Trypsin binding to the inner wall of the capillary was confirmed by confocal laser scanning microscopy. The properties of the trypsin-DNA conjugated, PAMAM-modified capillary microreactor were investigated by monitoring hydrolysis of Nα-benzoyl-L-arginine ethyl ester. Through the hybridization and dehybridization of the DNA, the inner wall of the capillary functionalized with trypsin can be regenerated, thus indicating the renewability of this enzyme microreactor. In addition, these results demonstrated that introduction of PAMAM enabled higher amounts of trypsin to be immobilized, markedly improving the enzymolysis efficiency, compared with traditional modified capillaries. The digestion performance of the trypsin capillary microreactor was further evaluated by digesting cytochrome C, and a peptide numbers of 8, and a sequence coverage of 59% were obtained. This renewable and efficient immobilized trypsin capillary microreactor combines advantages of both DDI technology and PAMAM, and is potentially adaptable to high-throughput enzyme assays in biochemical and clinical research.

Spatially Resolved Peptide-DNA Nanoassemblages for Biomarker Detection: A Synergy of DNA-Directed Immobilization and Nanografting.

Peptide microarrays are becoming a promising alternative to protein microarrays due to the challenges associated with protein immobilization and purification. Here, we put forward a novel experimental-based approach that combines DNA-directed immobilization, nanografting, and atomic force height measurements to immobilize computationally designed cyclic peptide on an ultra-flat gold substrate. This procedure yields peptide-DNA nanoarrays, which can bind to the solvent-exposed site on the Beta-2-microglobulin (ß2m).

High Activity and Convenient Ratio Control: DNA-Directed Coimmobilization of Multiple Enzymes on Multifunctionalized Magnetic Nanoparticles.

The development of new methods for fabricating artificial multienzyme systems has attracted much interest because of the potential applications and the urgent need for multienzyme catalysts. Controlling the enzyme ratio is critical for improving the cooperative enzymatic activity in multienzyme systems. Herein, we introduce a versatile strategy for fabricating a multienzyme system by coimmobilizing horseradish peroxidase (HRP) and glucose oxidase (GOx) on magnetic nanoparticles multifunctionalized with dopamine derivatives through DNA-directed immobilization. This multienzyme system exhibited precise enzyme ratio control, high catalytic efficiency, magnetic retrievability, and enhanced stability. The enzyme ratio was conveniently adjusted, as required, by regulating the quantity of functional groups on the multifunctionalized nanoparticles. The optimal mole ratio of GOx/HRP was 2:1. The Michaelis constant Km and specificity constant (kcat/Km, where kcat is the catalytic rate constant) of the multienzyme system were 1.41 mM and 5.02 s-1 mM-1, respectively, which were approximately twice the corresponding values of free GOx&HRP. The increased bioactivity of the multienzyme system was ascribed to the colocalization of the involved enzymes and the promotion of DNA-directed immobilization. Given the wide variety of possible enzyme associations and the high efficiency of this strategy, we believe that this work provides a new route for the fabrication of artificial multienzyme systems and can be extended for a wide range of applications in diagnosis, biomedical devices, and biotechnology.

Multimodal plasmonic biosensing nanostructures prepared by DNA-directed immobilization of multifunctional DNA-gold nanoparticles.

Biofunctional multimodal plasmonic nanostructures suitable for multiplexed localized surface plasmon resonance (LSPR) biosensing have been created by DNA-directed immobilization (DDI) of two distinct multifunctional biohybrid gold nanoparticles. Gold nanoparticles (AuNP) of distinct sizes, and therefore showing distinct plasmon resonant peaks (RP), have been biofunctionalized and codified with two different single stranded-DNA (ssDNA) chains. One of these oligonucleotide chains has been specifically designed to direct each AuNP to a distinct location of the surface of a DNA microarray chip through specific hybridization with complementary oligonucleotide strands. Scanning Electron Microscopy (SEM) has been used to demonstrate selective immobilization of each AuNP on distinct spots. The second ssDNA chain of the AuNPs provides the possibility to introduce by hybridization distinct types of bioactive molecules or bioreceptors, on a reversible manner. In this work, hapten-oligonucleotide bioconjugate probes, with sequences complementary to the second ssDNA linked to the AuNP, have been synthesized and used to create multiplexed hapten-biofuncionalized plasmonic nanostructures. The oligonucleotide probes consist on anabolic androgenic steroid haptens (AAS) covalently linked to specifically designed oligonucleotide sequences. The biofunctionality of these plasmonic nanostructures has been demonstrated by fluorescent microarray immunoassay and LSPR measurements, recording the shift of the RP produced after the antibody binding to the corresponding hapten-oligonucleotide probes immobilized on the nanostructured surface. Preliminary data show that this approach could allow manufacturing multifunctional multimodal LSPR chips for multiplexed analysis of different substances reaching very good detectability. Thus, small molecular weigh, analytes such as stanozolol (ST,) could be detected at concentrations in the low nM range. The results here presented open the door for an easy way to construct site-encoded multiplexed multimodal LSPR sensor transducers, combining the DDI strategies with multimodal biohybrid nanoparticles showing distinct optical properties.