Proximity-Dependent Activation of Split DNAzyme Kinase.

Binary (also known as split) nucleic acid enzymes have emerged as novel tools in biosensors. We report a new split strategy to split the DNAzyme kinase into two independent and non-functional fragments, denoted DK1sub and DK1enz. In the presence of the specific target, their free ends are brought sufficiently close to interact with each other without the formation of Watson-Crick base pairings between Dk1sub and Dk1enz, thus allowing the DNA phosphorylation reaction. We term this approach proximity-dependent activation of split DNAzyme kinase (ProxSDK). The utility of ProxSDK is demonstrated by engineering a biosensing system that is capable of measuring specific DNA-protein interactions. We envision that the approach described herein will find useful applications in biosensing, imaging, and clinical diagnosis.

Amplification-free detection of Escherichia coli using an acidic deoxyribozyme-based paper device.

We reported a colorimetric paper-based device by integrating the modified acid RNA-cleaving DNAzymes (MaRCD-EC1) for highly sensitive (detection limit = 102 CFU mL-1), and rapid (within 30 min) detection of E. coli without amplification. This device exhibited a clinical sensitivity of 100% and a specificity of 100% in identifying E. coli-associated urinary tract infections (UTIs) using the clinical urine samples.

Self-Assembly of Single-Virus SERS Hotspots for Highly Sensitive In Situ Detection of SARS-CoV-2 on Solid Surfaces.

Microbial surface transmission has aroused great attention since the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Developing a simple in situ detection method for viruses on solid surfaces is of great significance for timely public health surveillance. Taking advantage of the natural structure of SARS-CoV-2, we reported the assembly of Au@AgNPs on the surface of a single virus by the specific aptamer-spike protein interaction. Multiple hotspots can be created between the neighboring Au@AgNPs for the highly sensitive surface-enhanced Raman scattering (SERS) detection of SARS-CoV-2. Using two different aptamers labeled with Cy3 and Au@AgNPs, in situ SERS detection of pseudotyped SARS-CoV-2 (PSV) on packaging surfaces was achieved within 20 min, with a detection limit of 5.26 TCID50/mL. For the blind testing of 20 PSV-contaminated packaging samples, this SERS aptasensor had a sensitivity of 100% and an accuracy of 100%. This assay has been successfully applied to in situ detection of PSV on the surfaces of different packaging materials, suggesting its potential applicability.

A high-fidelity DNAzyme-assisted CRISPR/Cas13a system with single-nucleotide resolved specificity.

A CRISPR/Cas system represents an innovative tool for developing a new-generation biosensing and diagnostic strategy. However, the off-target issue (i.e., mistaken cleavage of nucleic acid targets and reporters) remains a great challenge for its practical applications. We hypothesize that this issue can be overcome by taking advantage of the site-specific cleavage ability of RNA-cleaving DNAzymes. To test this idea, we propose a DNAzyme Operation Enhances the Specificity of CRISPR/Cas13a strategy (termed DOES-CRISPR) to overcome the problem of relatively poor specificity that is typical of the traditional CRISPR/Cas13a system. The key to the design is that the partial hybridization of the CRISPR RNA (crRNA) with the cleavage fragment of off-target RNA was not able to activate the collateral cleavage activity of Cas13a. We showed that DOES-CRISPR can significantly improve the specificity of traditional CRISPR/Cas13a-based molecular detection by up to ∼43-fold. The broad utility of the strategy is illustrated through engineering three different systems for the detection of microRNAs (miR-17 and let-7e), CYP2C19*17 gene, SARS-Cov-2 variants (Gamma, Delta, and Omicron) and Omicron subtypes (BQ.1 and XBB.1) with single-nucleotide resolved specificity. Finally, clinical evaluation of this assay using 10 patient blood samples demonstrated a clinical sensitivity of 100% and specificity of 100% for genotyping CYP2C19*17, and analyzing 20 throat swab samples provided a diagnostic sensitivity of 95% and specificity of 100% for Omicron detection, and a clinical sensitivity of 92% and specificity of 100% for XBB.1 detection.

A DNAzymes-in-droplets assay for Burkholderia gladioli pathovar cocovenenans with single-bacterium sensitivity.

Foodborne pathogens pose a serious risk to human health, and the simple and rapid detection of such bacteria in complex food matrices remains challenging. Herein, we present the selection and characterization of a novel RNA-cleaving fluorogenic DNAzyme, named RFD-BC1, with exceptional specificity for Burkholderia gladioli pv. cocovenenans (B. cocovenenans), a pathogen strongly associated with fatal food poisoning cases. RFD-BC1 was activated by a protein secreted specifically by whole viable B. cocovenenans and displayed an optimum pH distinct from the selection pH, with a rate constant of approximately 0.01 min-1 at pH 5.0. Leveraging this newly discovered DNAzyme, we developed a novel system, termed DNAzymes-in-droplets (DID), that integrates droplet microfluidics to achieve the rapid and selective detection of live B. cocovenenans with single-cell sensitivity. We believe that the approach described herein holds promise for combating specific bacterial pathogens in food samples, offering significant potential for broader applications in food safety and public health.

In Vitro Selection of M2+-Independent, Fast-Responding Acidic Deoxyribozymes for Bacterial Detection.

We report on the first efforts to isolate acidic RNA-cleaving DNAzymes (aRCDs) from a random-sequence DNA pool by in vitro selection that are activated by a microbe Escherichia coli (E. coli), at pH 5.3. Importantly, these E. coli-responsive aRCDs only require monovalent metal ions as cofactors for cleaving a fluorogenic chimeric DNA/RNA substrate. Such characteristics can be used to efficiently protect RCDs from both intrinsic chemical instability and external enzymatic degradation. One remarkable DNAzyme, aRCD-EC1, is specific for E. coli, and its target is likely a protein. Furthermore, truncated aRCD-EC1 had significantly improved catalytic activity with an observed rate constant (kobs) of 1.18 min-1, making it the fastest bacteria-responding RCD reported to date. Clinical evaluation of this aRCD-based fluorescent assay using 40 patient urine samples demonstrated a diagnostic sensitivity of 100% and a specificity of 100% at a total analysis time of 50 min without a bacterial culture. This work can expand the repertoire of DNAzymes that are active under nonphysiological conditions, thus facilitating the development of diverse DNAzyme-based biosensors in clinical diagnosis.

Integration of CRISPR/Cas13a and V-Shape PCR for Rapid, Sensitive, and Specific Genotyping of CYP2C19 Gene Polymorphisms.

Rapid detection of single nucleotide polymorphisms (SNPs) in the CYP2C19 gene is of great significance for clopidogrel-accurate medicine. CRISPR/Cas systems have been increasingly used in SNP detection due to their single-nucleotide mismatch specificity. PCR, as a powerful amplification tool, has been incorporated into the CRISPR/Cas system to improve the sensitivity. However, the complicated three-step temperature control of the conventional PCR impeded rapid detection. The "V" shape PCR can shorten about 2/3 of the amplification time compared with conventional PCR. Herein, we present a new system termed the "V" shape PCR-coupled CRISPR/Cas13a (denoted as VPC) system, achieving the rapid, sensitive, and specific genotyping of CYP2C19 gene polymorphisms. The wild- and mutant-type alleles in CYP2C19*2, CYP2C19*3, and CYP2C19*17 genes can be discriminated by using the rationally programmed crRNA. A limit of detection (LOD) of 102 copies/µL was obtained within 45 min. In addition, the clinical applicability was demonstrated by genotyping SNPs in CYP2C19*2, CYP2C19*3, and CYP2C19*17 genes from clinical blood samples and buccal swabs within 1 h. Finally, we conducted the HPV16 and HPV18 detections to validate the generality of the VPC strategy.

Nucleic Acid Enzyme-Activated CRISPR-Cas12a With Circular CRISPR RNA for Biosensing.

clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are increasingly used in biosensor development. However, directly translating recognition events for non-nucleic acid targets by CRISPR into effective measurable signals represents an important ongoing challenge. Herein, it is hypothesized and confirmed that CRISPR RNAs (crRNAs) in a circular topology efficiently render Cas12a incapable of both site-specific double-stranded DNA cutting and nonspecific single-stranded DNA trans cleavage. Importantly, it is shown that nucleic acid enzymes (NAzymes) with RNA-cleaving activity can linearize the circular crRNAs, activating CRISPR-Cas12a functions. Using ligand-responsive ribozymes and DNAzymes as molecular recognition elements, it is demonstrated that target-triggered linearization of circular crRNAs offers great versatility for biosensing. This strategy is termed as "NAzyme-Activated CRISPR-Cas12a with Circular CRISPR RNA (NA3C)." Use of NA3C for clinical evaluation of urinary tract infections using an Escherichia coli-responsive RNA-cleaving DNAzyme to test 40 patient urine samples, providing a diagnostic sensitivity of 100% and specificity of 90%, is further demonstrated.

Functional DNA Superstructures Exhibit Positive Homotropic Allostery in Ligand Binding.

Inspired by intrinsically disordered proteins in nature, DNA aptamers can be engineered to display strongly homotropic allosteric (or cooperative) ligand binding, representing a unique feature that could be of great utility in applications such as biosensing, imaging and drug delivery. The use of an intrinsic disorder mechanism, however, comes with an inherent drawback of significantly reduced overall binding affinity. We hypothesize that it could be addressed via the design of multivalent supramolecular aptamers. We built functional DNA superstructures (denoted as 3D DNA), made of long-chain DNA containing tandem repeating DNA aptamers (or concatemeric aptamers). The 3D DNA systems exhibit highly cooperative binding to both small molecules and proteins, without loss of binding affinities of their parent aptamers. We further produced a highly responsive sensor for fluorescence imaging of glutamate stimulation-evoked adenosine triphosphate (ATP) release in neurons, as well as force stimulus-triggered ATP release in astrocytes.

Self-assembly of protein-DNA superstructures for alkaline phosphatase detection in blood.

We designed a paper-based analytical device by integrating horseradish peroxidase (HRP)-encapsulated 3D DNA for visual detection of alkaline phosphatase (ALP). This device allows on-paper sample pre-treatment, target recognition and signal readout, enabling simple (without additional pre-treatment of blood samples) and rapid (within 23 min) determination of ALP in clinical samples.

Engineering a Ligase Binding DNA Aptamer into a Templating DNA Scaffold to Guide the Selective Synthesis of Circular DNAzymes and DNA Aptamers.

Functional nucleic acids (FNAs), such as DNAzymes and DNA aptamers, can be engineered into circular forms for improved performance. Circular FNAs are promising candidates for bioanalytical and biomedical applications due to their intriguing properties of enhanced biological stability and compatibility with rolling circle amplification. They are typically made from linear single-stranded (ss) DNA molecules via ligase-mediated ligation. However, it remains a great challenge to synthesize circular ssDNA molecules in high yield due to inherent side reactions where two or more of the same ssDNA molecules are ligated. Herein, we present a strategy to overcome this issue by first using in vitro selection to search from a random-sequence DNA library a ligatable DNA aptamer that binds a DNA ligase and then by engineering this aptamer into a general-purpose templating DNA scaffold to guide the ligase to execute selective intramolecular circularization. We demonstrate the broad utility of this approach via the creation of several species of circular DNA molecules, including a circular DNAzyme sensor for a bacterium and a circular DNA aptamer sensor for a protein target with excellent detection sensitivity and specificity.

Total Bioaerosol Detection by Split Aptamer-Based Electrochemical Nanosensor Chips.

Bioaerosols could carry and spread harmful microorganisms, thus posing a continuous threat to human beings and livestock health. Early warning and management are crucial for controlling the spread of bioaerosols. Herein, we developed a split aptamer (SA)-based electrochemical nanosensor chip (denoted SAE-nChip) for rapid and sensitive detection of adenosine triphosphate (ATP) in bioaerosols. The platform features two components: split DNA aptamers for their ability to bind ATP and undergo target-induced assembly on the chip surface and ZIF-8@MXene composites for their ability to provide a high surface density of aptamer-binding sites and facilitate the electron transfer at the biointerface. The SAE-nChip was capable of detecting ATP with a detection limit of 10 pM. Furthermore, this assay allowed the detection of ATP in cultured microorganisms and collected real bioaerosols. Overall, this strategy of interfacing DNA aptamers with MXene-based composite materials represents a versatile approach for the ubiquitous detection of biochemical targets in bioaerosols.

Colorimetric Detection of DNase Type I 3'OH DNA Ends Using an Isothermal Amplification-Assisted Paper-Based Analytical Device.

The generation of DNase type I 3'OH DNA ends is closely related to the harm of endogenous reactive oxygen species (ROS) and environmental genotoxic agents. The evaluation of this type of DNA damage plays an important role in clinical intervention and environmental toxicity assessment. Terminal deoxynucleotidyl transferase (TdT)-assisted isothermal amplification (TAIA) offers a facile and versatile way to detect DNase type I 3'OH DNA ends. Its ability of templated-independent isothermal amplification is one unique feature. Here, we reported a paper-based analytical device (PAD) coupled with a smartphone for the detection of DNase type I 3'OH DNA ends using TAIA and colorimetric signal readout. We achieved the integration of cell lysis, DNA extraction, TAIA, horseradish peroxidase (HRP)-enabled colorimetric reaction, and signal readout. This device could achieve a limit of detection of 264 cells with a total assay time of less than 45 min. By combining PAD with a smartphone, the integrated platform could be used for the visual and quantitative analysis of DNA damages with the advantages of ease-to-use, fast response, inexpensive, and instrument free. Furthermore, successful assessment of the genotoxicity in wastewater effluents suggested the great promise of the integrated platform for on-site testing in practical applications.

An Electrochemical Aptasensor Integrating Zeolitic Imidazolate Framework for Highly Selective Detection of Bioaerosols.

Bioaerosols are the biological materials in the air, which may cause a continuous threat to human health. However, there are many challenges in monitoring bioaerosols such as lack of sensitivity and selectivity. Herein, we synthesized a series of nanohybrids containing zeolitic imidazolate frameworks (ZIFs) and covalent organic frameworks (COFs) to construct an electrochemical aptasensor for detecting adenosine triphosphate (ATP), a biomarker for bioaerosols. The synthesized nanohybrids can not only improve the selectivity of aptasensor because of the original crystal and chemical features of ZIF-67, but also boost its sensitivity due to the excellent conductivity of COFs. After optimizing the nanohybrids, the novel developed sensing platform achieved highly selective detection of ATP with an excellent detection limit of 0.11 nM in a wide linear range from 0.1 nM to 100 nM. Furthermore, this assay was applied to detect bioaerosols in real air samples, and the result showed a positive correlation with that of the culturing-based method, suggesting its potential applicability.

Quantifying DNA damage on paper sensors via controlled template-independent DNA polymerization.

We report on a paper-based sensor capable of performing template-independent DNA synthesis by terminal deoxynucleotidyl transferase (TdT). Importantly, we observed that TdT efficiently incorporates fluorescently labeled dUTP on to 3'-OH ends of DNA strands in a strictly controllable manner on cellulose paper, in comparison to its distributive mode of DNA synthesis in solution. Due to the high roughness and porous nature of cellulose paper, we attribute this controllable DNA polymerization to the pore confinement effect on the catalytic behaviour of TdT. Taking advantage of this finding, we proposed a paper-assisted TdT (PAT) assay for absolute quantification of alkylated DNA lesions (N7-methylguanine), DNA deamination (cytosine-to-uracil) and DNA oxidation (8-oxo-7,8-dihydroguanine) by combining various DNA glycosylases. This PAT assay provides a low-cost, high throughput and easy to use method for quantifying the absolute levels of various types of DNA lesions, thus making it well-suited for drug development, genotoxicity testing, and environmental toxicology.

Single bacteria detection by droplet DNAzyme-coupled rolling circle amplification.

We described a new system termed droplet DNAzyme-coupled rolling circle amplification (dDRCA) that can selectively detect bacteria from clinical urine samples with single-cell sensitivity within 1.5 h compared with the several hours needed for traditionally used culture-based methods.

Gas sensing based on metal-organic frameworks: Concepts, functions, and developments.

Effective detection of pollutant gases is vital for protection of natural environment and human health. There is an increasing demand for sensing devices that are equipped with high sensitivity, fast response/recovery speed, and remarkable selectivity. Particularly, attention is given to the designability of sensing materials with porous structures. Among diverse kinds of porous materials, metal-organic frameworks (MOFs) exhibit high porosity, high degree of crystallinity and exceptional chemical activity. Their strong host-guest interactions with guest molecules facilitate the application of MOFs in adsorption, catalysis and sensing systems. In particular, the tailorable framework/composition and potential for post-synthetic modification of MOFs endow them with widely promising application in gas sensing devices. In this review, we outlined the fundamental aspects and applications of MOFs for gas sensors, and discussed various techniques of monitoring gases based on MOFs as functional materials. Insights and perspectives for further challenges faced by MOFs are discussed in the end.

Investigation of interaction between MXene nanosheets and human plasma and protein corona composition.

The composition of protein corona affects the behavior and fate of nanoparticles in biological systems, which strongly relates to the intrinsic properties of nanoparticles and proteins. Here, three types of MXene Ti3C2Tx nanosheets are prepared by different etching methods, and certain physicochemical characteristics of the nanosheets before and after exposure to human plasma (HP) are characterized. The Ti3C2Tx nanosheets with protein coronas suffer more easily from aggregation than pristine Ti3C2Tx. The composition of protein coronas by LC-MS/MS-based label-free proteomic analysis reveals a high overlap of protein types and functions but a significant difference in relative protein abundance for the three Ti3C2Tx. Immunoglobulins and coagulation proteins are highly enriched while albumin is depleted in the coronas compared with their abundance in original HP. The random forest classification model predicts that the main driving forces for the adsorption of HP proteins on Ti3C2Tx are hydrogen bonding, steric hindrance, and hydrophobic interaction. This study provides insights into the colloidal stability of Ti3C2Tx nanosheets and their interaction with human plasma proteins.

Siderophores provoke extracellular superoxide production by Arthrobacter strains during carbon sources-level fluctuation.

Superoxide and other reactive oxygen species (ROS) shape microbial communities and drive the transformation of metals and inorganic/organic matter. Taxonomically diverse bacteria and phytoplankton produce extracellular superoxide during laboratory cultivation. Understanding the physiological reasons for extracellular superoxide production by aerobes in the environment is a crucial question yet not fully solved. Here, we showed that iron-starving Arthrobacter sp. QXT-31 (A. QXT-31) secreted a type of siderophore [deferoxamine (DFO)], which provoked extracellular superoxide production by A. QXT-31 during carbon sources-level fluctuation. Several other siderophores also demonstrated similar effects to A. QXT-31. RNA-Seq data hinted that DFO stripped iron from iron-bearing proteins in electron transfer chain (ETC) of metabolically active A. QXT-31, resulting in electron leakage from the electron-rich (resulting from carbon sources metabolism by A. QXT-31) ETC and superoxide production. Considering that most aerobes secrete siderophore(s) and undergo carbon sources-level fluctuation, the superoxide-generation pathway is likely a common pathway by which aerobes produce extracellular superoxide in the environment, thus influencing the microbial community and cycling of elements. Our results pointed that the ubiquitous siderophore might be the potential driving force for the microbial generation of superoxide and other ROS and revealed the important role of iron physiology in microbial ROS generation.

An origami paper-based analytical device for DNA damage analysis.

Detection and characterization of DNA damage plays a critical role in genotoxicity testing, drug screening, and environmental health. We developed a fully integrated origami paper-based analytical device (oPAD) for measuring DNA damage. This simple device allows on-paper cell lysis, DNA extraction, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction and signal readout with simple operation steps, enabling rapid (within 30 min) and high throughput assessment of multiple DNA damages induced by exogenous chemical agents.