Enhanced sensitivity in Staphylococcus aureus detection: Unveiling the impact of lipid composition on the performance of carboxyfluorescein (CF)-Loaded liposome-based assay.

Liposomes have emerged as versatile nanocarriers, finding applications not only in drug delivery but also in pathogen detection and diagnostics. This study aimed to enhance the sensitivity of liposomes to Staphylococcus aureus by investigating the impact of lipid composition on liposomes loaded with 5(6)-carboxyfluorescein (CF). Liposomes were fabricated using various concentrations of cholesterol (10-40 mol%) combined with saturated phospholipids. Dynamic light scattering results revealed that higher cholesterol concentrations led to reduced liposome size, CF release (%), and entrapment efficiency (%). Liposome sensitivity towards S. aureus was evaluated by using CF-loaded liposomes with and without aptamer insertion. Liposomes with a higher cholesterol content (40 mol%) exhibited a strong ability to detect low bacterial concentrations down to 5 × 102 CFU/mL without relying solely on specific receptor-ligand recognition. However, functionalizing the liposome with an aptamer further improved the specificity and sensitivity of S. aureus detection at even lower concentrations, down to 80 CFU/mL, in the wide range of 80-107 CFU/mL. This study highlights the potential for optimizing the lipid composition of liposomes to improve their sensitivity for pathogen detection, particularly when combined with aptamer-based strategies.

Systematic bio-fabrication of aptamers and their applications in engineering biology.

Aptamers are single-stranded DNA or RNA molecules that have high affinity and selectivity to bind to specific targets. Compared to antibodies, aptamers are easy to in vitro synthesize with low cost, and exhibit excellent thermal stability and programmability. With these features, aptamers have been widely used in biology and medicine-related fields. In the meantime, a variety of systematic evolution of ligands by exponential enrichment (SELEX) technologies have been developed to screen aptamers for various targets. According to the characteristics of targets, customizing appropriate SELEX technology and post-SELEX optimization helps to obtain ideal aptamers with high affinity and specificity. In this review, we first summarize the latest research on the systematic bio-fabrication of aptamers, including various SELEX technologies, post-SELEX optimization, and aptamer modification technology. These procedures not only help to gain the aptamer sequences but also provide insights into the relationship between structure and function of the aptamers. The latter provides a new perspective for the systems bio-fabrication of aptamers. Furthermore, on this basis, we review the applications of aptamers, particularly in the fields of engineering biology, including industrial biotechnology, medical and health engineering, and environmental and food safety monitoring. And the encountered challenges and prospects are discussed, providing an outlook for the future development of aptamers.

Nanosensor Based on the Dual-Entropy-Driven Modulation Strategy for Intracellular Detection of MicroRNA.

The entropy-driven strategy has been proposed as a milestone work in the development of nucleic acid amplification technology. With the characteristics of an enzyme-free, isothermal, and relatively simple design, it has been widely used in the field of biological analysis. However, it is still a challenge to apply entropy-driven amplification for intracellular target analysis. In this study, a dual-entropy-driven amplification system constructed on the surface of gold nanoparticles (AuNPs) is developed to achieve fluorescence determination and intracellular imaging of microRNA-21 (miRNA-21). The dual-entropy-driven amplification strategy internalizes the fuel chain to avoid the complexity of the extra addition in the traditional entropy-driven amplification strategy. The unique self-locked fuel chain system is established by attaching the three-stranded structure on two groups of AuNPs, where the Cy5 fluorescent label was first quenched by AuNPs. After the target miRNA-21 is identified, the fuel chain will be automatically unlocked, and the cycle reaction will be driven, leading to fluorescence recovery. The self-powered and waste-recycled fuel chain greatly improves the automation and intelligence of the reaction process. Under the optimal conditions, the linear response range of the nanosensor ranges from 5 pM to 25 nM. This nanoreaction system can be used to realize intracellular imaging of miRNA-21, and its good specificity enables it to distinguish tumor cells from healthy cells. The development of the dual-entropy-driven strategy provides an integrated and powerful way for intracellular miRNA analysis and shows great potential in the biomedical field.

Quantitative Determination of Staphylococcus aureus Using Aptamer-Based Recognition and DNA Amplification Machinery.

Staphylococcus aureus (S. aureus) is a common foodborne pathogen that threatens human health and safety. It is significant to develop sensitive detection methods for the monitoring of S. aureus contamination in food and environment. Herein, a novel machinery based on aptamer recognition, DNA walker, and rolling circle amplification (RCA) was designed, which can form unique DNA nanoflower and subsequently detect low-level S. aureus contamination in samples. To this end, two rationally designed DNA duplexes were modified on the surface of the electrode to identify S. aureus through the high affinity between aptamers and S. aureus. Combined with the repeated movement of DNA walker machinery on the electrode surface and RCA technology, a unique DNA nanoflower structure was formed. This can effectively transform the biological information of aptamer recognition of S. aureus into a significantly amplified electrochemical signal. Through reasonable design and optimization of the parameters of each part, the linear response range of the S. aureus biosensor is from 60 to 6 × 107 CFU/mL and the detection limit is as low as 9 CFU/mL.

A pH-Gated Functionalized Hollow Mesoporous Silica Delivery System for Photodynamic Sterilization in Staphylococcus aureus Biofilm.

Multidrug-resistant bacteria are increasing, particularly those embedded in microbial biofilm. These bacteria account for most microbial infections in humans. Traditional antibiotic treatment has low efficiency in sterilization of biofilm-associated pathogens, and thus the development of new approaches is highly desired. In this study, amino-modified hollow mesoporous silica nanoparticles (AHMSN) were synthesized and used as the carrier to load natural photosensitizer curcumin (Cur). Then glutaraldehyde (GA) and polyethyleneimine (PEI) were used to seal the porous structure of AHMSN by the Schiff base reaction, forming positively charged AHMSN@GA@PEI@Cur. The Cur delivery system can smoothly diffuse into the negatively charged biofilm of Staphylococcus aureus (S. aureus). Then Cur can be released to the biofilm after the pH-gated cleavage of the Schiff base bond in the slightly acidic environment of the biofilm. After the release of the photosensitizer, the biofilm was irradiated by the blue LED light at a wavelength of 450 nm and a power of 37.4 mV/cm2 for 5 min. Compared with the control group, the number of viable bacteria in the biofilm was reduced by 98.20%. Therefore, the constructed pH-gated photosensitizer delivery system can efficiently target biofilm-associated pathogens and be used for photodynamic sterilization, without the production of antibiotic resistance.

A portable and quantitative detection of microRNA-21 based on cascade enzymatic reactions with dual signal outputs.

MicroRNAs (miRNAs) are physiological status-related molecules which can be used as biomarkers for diseases, such as cancers. The point-of-care testing (POCT) of miRNAs has great application potential in early diagnosis and process monitoring of diseases. In this paper, a fast and dual signal outputs detection for microRNA-21 (miRNA-21) was established by using both personal glucose meter (PGM) and fluorescence spectrometer. In such an assay protocol, a dual-functional hairpin structure was rationally designed to recognize miRNA-21 and serve as the carrier of the reporter adenosine monophosphate (AMP). The hairpin structure can be specifically degraded by exonuclease T (Exo T) after hybridization with the target miRNA-21, releasing a large amount of AMP as the reporter. Then a smart signal conversion machinery composed of four enzymes and the corresponding substrates was employed to produce dual output signals through enzymatic cascade reactions. The machinery includes two parts: an adenosine triphosphate (ATP) generation system and a glucose consumption/NADPH production system. The produced AMP in the former step triggers the production of ATP, and subsequently the consumption of glucose and the production of NADPH. The changes of both glucose and NADPH are proportional to the concentration of miRNA-21, and can be determined by PGM and fluorescence spectrometer, respectively. Besides, the build-in substrate-recycling mechanism achieves signal amplification of the cascade enzymatic reactions. Under the optimal experimental conditions, the PGM signal is linearly correlated with the concentration of miRNA-21 in the range from 5 to 150 nM, with the limit of detection (LOD) of 3.65 nM. The LOD of fluorescence detection mode is even lowered to 0.03 nM. The miRNA-21-spiked serum samples, as well as the actual serum samples from cancer patients, have been successfully detected by this detection strategy. Thus the established assay provides a POCT solution for cancer diagnosis and prognosis.

Development of a sensitive and stable chemiluminescent immunoassay for detection of birch pollen allergic specific IgE based on recombinant Bet v1 protein.

Background Quantitative detection of allergens is of great significance for clarifying the cause, treatment, and prevention of allergy disease. Birch pollen is one of the most common inhalational allergens and Bet v1 is the major component allergen of birch allergen. This study aims to develop a stable and sensitive chemiluminescence immunoassay (CLIA) for the detection of birch pollen allergic specific IgE (sIgE) based on recombinant Bet v1 (rBet v1) protein. Methods rBet v1 protein was expressed in Escherichia coli and purified. Then rBet v1 was applied to detect sIgE in human serum. The performance of the established CLIA was evaluated and compared with Phadia rBet v1 fluorescence enzyme immunoassay (FEIA) system. Results The developed CLIA for sIgE to rBet v1 detection shows excellent performance. The assay showed a linear range from 0.1 to 100 IU/mL, with a low detection limit of 0.06 IU/mL. A total of 164 samples were evaluated by CLIA and compared with the results of FEIA. The positive, negative, and total coincidence rate was 90.6% (87/96), 91.2% (62/68), and 90.9% (149/164), respectively. The r-value of Spearman's rank correlation analysis was 0.935 (P < 0.001). The use of high levels of bilirubin (50 mg/dL), hemoglobin (400 mg/dL) and lipid (2000 mg/dL) didn't interfere with the results. Conclusions The proposed CLIA exhibits excellent performance for the detection of rBet v1 specific IgE. It can be a reliable tool for the early diagnosis of hypersensitivity.

An ultrasensitive biosensor for dual-specific DNA based on deposition of polyaniline on a self-assembled multi-functional DNA hexahedral-nanostructure.

Kras and Braf are major oncogenes. The mutation of Kras codon 12 or Braf V600E can lead to ovarian carcinoma. The detection of oncogene-related DNAs and their mutations offers solution for early diagnosis of ovarian cancer. Herein, a size-tunable multi-functional DNA hexahedral-nanostructure (DHN) has been rationally designed and modified on the electrode to response to Kras and Braf DNA. The size of DHN is controlled via polyadenines (polyA). The complete self-assembly of DHN depends on the presence of both target DNAs and two assistant probes. Meanwhile, a HRP-mimicking DNAzyme forms in DHN, which catalyzes the polymerization of aniline. The produced polyaniline is utilized as the output signal through differential pulse voltammetry (DPV). The biosensor shows the linear range from 100 fM to 1 µM, with the detection limit of 48.7 fM for Kras gene; and the linear range from 100 fM to 100 nM, with the detection limit of 44.1 fM for Braf gene, respectively. Since the current response depends on both gene sequences, the high specificity of the biosensor endows it to operate in an "OR"-type logic gate to discriminate the mutation of both genes. When Kras codon 12 or Braf V600E mutation happens, the response decreases significantly due to the incomplete formation of DNAzyme in DHN. The practicability of the biosensor has been verified through challenging human serum samples. Thus, it has great potential for clinical diagnosis of ovarian cancer through simultaneous detection of Kras and Braf genes and their mutations.

Self-Assembled DNA Nanoflowers Triggered by a DNA Walker for Highly Sensitive Electrochemical Detection of Staphylococcus aureus.

With the development of DNA nanotechnology, DNA has been widely used to construct a variety of nanomachines. Among them, a DNA walker is a unique nanomachine that can move continuously along a specific orbit to fulfill diverse functions. In this paper, a dual signal amplification electrochemical biosensor based on a DNA walker and DNA nanoflowers is constructed for high sensitivity detection of Staphylococcus aureus (S. aureus). Two groups of double-stranded DNA are modified on the surface of a gold electrode. The binding of S. aureus with its aptamer induces the disintegration of the long double strands and releases the DNA walker. With the help of exonuclease III (Exo III), the DNA walker moves along the electrode surface and continuously hydrolyzes the anchored short double strands. The introduction of a specially customized circular DNA and phi29 DNA polymerase initiates the rolling circle amplification (RCA) reaction. DNA nanoflowers are formed at high local concentration of DNA in the solution, which provide binding sites for electroactive methylene blue (MB) and thus produce intense signal. Under the best conditions, the current response is linearly related to the logarithm of the concentration of S. aureus ranging from 60 to 6 × 107 CFU/mL, and the detection limit is 9 CFU/mL. In addition, the proposed biosensor has achieved satisfactory results in the detection of actual water samples and diluted honey samples, which confirm the practicability of the biosensor and its application potential in environmental monitoring and food safety.

An electrochemical aptasensor for ATP based on a configuration-switchable tetrahedral DNA nanostructure.

A novel electrochemical aptasensor for ATP was developed based on an aptamer-embedded configuration-switchable tetrahedral DNA nanostructure (TDN) and the formation of a G-quadruplex. This unique TDN was formed through the self-assembly of four specially designed single-stranded DNA (ssDNA) sequences (S1, S2, S3 and S4). The TDN was immobilized on the surface of a Au electrode through the thiol groups at the 5'-end of S1, S2 and S3. Five edges of the TDN were designed to form a double helix to preserve the structural robustness of the tetrahedron, while the ATP aptamer embedded sequence (S3) was designed to be located at the rest edge. The two terminals of S4 at the same edge were composed of two split G-quadruplex-forming sequences, which were non-complementary to the aptamer. This edge offered the configuration-switchable characteristic of the TDN. In the absence of ATP, the TDN remained in a relaxed state, and the G-quadruplex cannot form due to the large distance between the split G-quadruplex-forming sequences. However, in the presence of ATP, the aptamer combined with ATP and shortened the distance between the split sequences, resulting in the taut state of the TDN and the formation of a G-quadruplex at the edge. After the addition of hemin, the differential pulse voltammograms (DPVs) were used to quantify ATP. The sensor revealed a dynamic response range from 0.1 nM to 1 µM, with a detection limit of 50 pM. In addition, the specificity and practicability in real samples were also verified, indicating its potential applications.

Screening and application of a truncated aptamer for high-sensitive fluorescent detection of metronidazole.

Aptamer-based biosensors have been widely constructed and applied to detect diverse targets. Metronidazole is a widely used broad-spectrum antibacterial drug, whose residue has multiple risks to human health. Herein, metronidazole-specific aptamers were selected from a random ssDNA library with the full length of 79 nucleotides (nt) based on DNA library-immobilized magnetic beads SELEX technology. After ten rounds of selection, four aptamers with highly similar secondary structures were selected, among which AP32, with the lowest dissociation constants, was chosen as the optimal aptamer for further optimization. Then a semi-rational post-SELEX truncation was carried out based on the secondary structure analysis and molecular docking, as well as affinity assessment. Redundant nucleotides in AP32 were stepwise removed without the decrease of affinity. Following such strategy, a truncated aptamer AP32-4 with the length of only 15 nt was eventually screened. The dissociation constant of 77.22 ± 11.27 nM is almost equivalent to the original AP32. Furthermore, an aptamer-based fluorescent biosensor for metronidazole was constructed based on AP32-4. With the help of exonuclease-assisted target-recycling amplification, the biosensor exhibits a linear detection range of 25-800 nM, and the detection limit of 10.50 nM. The biosensor was applied to detect metronidazole in honey samples. The results show that not only an efficient strategy for screening robust and practicable aptamers, but also an ultrahigh sensitive detection platform for metronidazole were established.

A fluorescent aptasensor for Staphylococcus aureus based on strand displacement amplification and self-assembled DNA hexagonal structure.

A fluorescent aptasensor for Staphylococcus aureus (S. aureus) is designed, which takes advantage of strand displacement amplification (SDA) technology and unique self-assembled DNA hexagonal structure. In the presence of S. aureus, a partially complementary strand of S. aureus aptamer (cDNA) is competitively released from cDNA/aptamer duplex immobilized on magnetic beads due to the affinity of the aptamer for S. aureus. The addition of primer starts the SDA reaction. With the catalysis of Bsm DNA polymerase and Nb.bpu10I endonuclease, a large number of single-stranded DNA (ssDNA) is produced, which induces the opening of a hairpin probe and the subsequent self-assembly to form a hexagonal structure. The staining of the DNA hexagon with SYBR Green I excites the fluorescence signal, which is used for detection. The aptasensor exhibits a broad linear range from 7 to 7 × 107 CFU/mL, with a detection limit of 1.7 CFU/mL for S. aureus. The sensor shows negligible responses to other bacteria. Moreover, the aptasensor has been applied to detect S. aureus in milk samples, and the results demonstrate the general applicability of the sensor and its prospect in systematic detection of S. aureus in food safety control and medicine-related fields. Graphical abstract The presence of S. aureus can be converted to the formation of unique DNA hexagonal structure and subsequent fluorescent signal by the combination of SDA with self-assembly technology.

Screening, Post-SELEX Optimization and Application of DNA Aptamers Specific for Tobramycin.

Tobramycin (TOB) is an aminoglycoside antibiotic. The residue of TOB in animal-derived foods and environment will be harmful to human health, and therefore the specific detection of TOB residue in food and water is of great importance. Herein, through magnetic beads-based SELEX, overall 37 ssDNA aptamers specific for TOB were screened after ten rounds of selection. The affinity and specificity of these aptamers were evaluated, among which No. 32 aptamer (Ap 32) exhibits excellent performance. Then a post-SELEX optimization of Ap 32 was carried out based on rational design, through which a truncated aptamer with the length of 34 nucleotides (Ap 32-2) was identified as the best aptamer for TOB. Finally, the application of the screened aptamer was explored. A colorimetric assay of TOB was established based on the aptamer-modified gold nanoparticles (AuNPs). In the range from 100 to 1400 nM, the absorbance of AuNPs solution at 520 nm was linearly decreased with the increased concentration of TOB. The detection limit was estimated to be 37.9 nM. The assay was applied to detect TOB residue in honey samples.

Switchable DNA tweezer and G-quadruplex nanostructures for ultrasensitive voltammetric determination of the K-ras gene fragment.

Voltammetric detection of the K-ras gene fragment was accomplished through the combined application of (a) a switchable DNA nanostructure, (b) the use of hairpin probe and exonuclease III (Exo III)-assisted signal amplification, (c) a split G-quadruplex, and (d) by exploiting the redox activity of DNAzyme. Three assistant oligonucleotides were designed to construct a DNA tweezer on a gold electrode. It is in "open state" in the absence of K-ras DNA. Then, a hairpin probe was introduced, whose stem-loop structure can be opened through hybridization with the K-ras DNA. Exo III is added which hydrolyzes the complementary region of the hairpin sequence to release a single-stranded rest fragment. The ssDNA hybridizes with the DNA tweezer on the electrode which thereby is switched to the "closed state". This leads to the formation of G-quadruplex due to the shortened distance of the split G-quadruplex-forming sequences in the tweezer. The voltammetric signal of the G-quadruplex-hemin complex, with a peak near -0.3 V vs. Ag/AgCl, is used as the signal output. Under the optimal conditions, the current response in differential pulse voltammetry (DPV) increases linearly with the concentration of K-ras DNA in the range of 0.01-1000 pM, and the detection limit is 2.4 fM. The assay can clearly discriminate K-ras DNA from a single-base mutation. The method has excellent selectivity and was applied to the determination of K-ras DNA in (spiked) serum samples. Graphical abstractSchematic representation of a method for the determination of the K-ras gene fragment through a combination of switchable DNA tweezer, split G-quadruplex, and exonuclease III (ExoIII)-assisted target recycling signal amplification.

Functional chimera aptamer and molecular beacon based fluorescent detection of Staphylococcus aureus with strand displacement-target recycling amplification.

A fluorescent detection of Staphylococcus aureus (S. aureus) is established based on a finely designed functional chimera sequence, a molecular beacon (MB), and strand displacement target recycling. The chimera sequence, which consists of the aptamer sequence of S. aureus and the complementary sequence of MB, can form a hairpin structure due to the existence of intramolecular complementary regions. When S. aureus is present, it binds to the aptamer region of the chimera, opens the hairpin and unlocks the complementary sequence of MB. Subsequently, the MB is opened and intensive fluorescence signal is restored. To increase the sensitivity of the detection, signal amplification is achieved through strand displacement-based target recycling. With the catalysis of Nb. Bpu10I nicking endonuclease and Bsm DNA polymerase, the MB sequence can be cleaved and then elongated to form a complete duplex with the chimera, during which S. aureus is displaced from the chimera and proceeded to the next round of the reaction. This assay displays a linear correlation between the fluorescence intensity and the logarithm of the concentration of S. aureus within a broad concentration range from 80 CFU/mL to 8 × 106 CFU/mL. The detection limit of 39 CFU/mL can be derived. The assay was applied to detect S. aureus in different water samples, and satisfactory recovery and repeatability were achieved. Hence the designed chimera sequence and established assay have potential application in environmental pollution monitoring.