High-affinity truncated aptamers for detection of Cronobacter spp with magnetic separation-assisted DNAzyme-driven 3D DNA walker.

After optimizing the original aptamer sequence by truncation strategy, a magnetic separation-assisted DNAzyme-driven 3D DNA walker fluorescent aptasensor was developed for detecting the food-borne pathogen Cronobacter species. Iron oxide magnetic nanoparticles (MNPs) modified with a hybrid of truncated aptamer probe and DNAzyme strand (AP-E1) denoted as MNPs@AP-E1, were employed as capture probes. Simultaneously, a DNAzyme-driven 3D-DNA walker was utilized as the signal amplification element. The substrate strand (Sub) was conjugated with the gold nanoparticles (AuNPs), resulting in the formation of AuNPs@Sub, which served as a 3D walking track. In the presence of the target bacteria and Mg2+, E1-DNAzyme was activated and moved along AuNPs@Sub, continuously releasing the signal probe. Under optimized conditions, a strong linear correlation was observed for Cronobacter sakazakii (C. sakazakii) in the concentration range 101 to 106 CFU mL-1, with a low detection limit of 2 CFU mL-1. The fluorescence signal responses for different Cronobacter species exhibited insignificant differences, with a relative standard deviation of 3.6%. Moreover, the aptasensor was successfully applied to determine  C. sakazakii in real samples with recoveries of 92.86%-108.33%. Therefore, the novel method could be a good candidate for ultra-sensitive and selective detection of Cronobacter species without complex manipulation.

Cascade DNA Circuits Mediated CRISPR-Cas12a Fluorescent Aptasensor based on Multifunctional Fe3 O4 @hollow-TiO2 @MoS2 Nanochains for Tetracycline Determination.

Herein, for the first time, the CRISPR-Cas12a system is combined with aptamer, cascaded dynamic DNA network circuits, and Fe3 O4 @hollow-TiO2 @MoS2 nanochains (Fe3 O4 @h-TiO2 @MoS2 NCs) to construct an efficient sensing platform for tetracycline (TC) analysis. In this strategy, specific recognition of the target is transduced and amplified into H1-H2 duplexes containing the specific sequence of Cas12a-crRNA through an aptamer recognition module and the dual amplification dynamic DNA network. Subsequently, the obtained activated Cas12a protein non-specifically cleaves the adjacent reporter gene ssDNA-FAM to dissociate the FAM molecule from the quencher Fe3 O4 @h-TiO2 @MoS2 NCs, resulting in the recovery of the fluorescence signal and further signal amplification. Particularly, the synthesized multifunctional Fe3 O4 @h-TiO2 @MoS2 NCs composites also exhibit superb magnetic separability and photocatalytic degradation ability. Under optimal conditions, the aptasensor displays a distinct linear relationship with the logarithm of TC concentration, and the limit of detection is as low as 0.384 pg mL-1 . Furthermore, the results of spiked recovery confirm the viability of the proposed aptasensor for TC quantification in real samples. This study extends the application of the CRISPR-Cas12a system in the field of analytical sensing and contributes new insights into the exploration of reliable tools for monitoring and treating hazards in food and environment.

Split aptamer acquisition mechanisms and current application in antibiotics detection: a short review.

Antibiotic contamination is becoming a prominent global issue. Therefore, sensitive, specific and simple technology is desirable the demand for antibiotics detection. Biosensors based on split aptamer has gradually attracted extensive attention for antibiotic detection due to its higher sensitivity, lower cost, false positive/negative avoidance and flexibility in sensor design. Although many of the reported split aptamers are antibiotics aptamers, the acquisition and mechanism of splitting is still unknow. In this review, six reported split aptamers in antibiotics are outlined, including Enrofloxacin, Kanamycin, Tetracycline, Tobramycin, Neomycin, Streptomycin, which have contributed to promote interest, awareness and thoughts into this emerging research field. The study introduced the pros and cons of split aptamers, summarized the assembly principle of split aptamer and discussed the intermolecular binding of antibiotic-aptamer complexes. In addition, the recent application of split aptamers in antibiotic detection are introduced. Split aptamers have a promising future in the design and development of biosensors for antibiotic detection in food and other field. The development of the antibiotic split aptamer meets many challenges including mechanism discovery, stability improvement and new biosensor development. It is believed that split aptamer could be a powerful molecular probe and plays an important role in aptamer biosensor.

Advances in riboswitch-based biosensor as food samples detection tool.

Food safety has always been a hot issue of social concern, and biosensing has been widely used in the field of food safety detection. Compared with traditional aptamer-based biosensors, aptamer-based riboswitch biosensing represents higher precision and programmability. A riboswitch is an elegant example of controlling gene expression, where the target is coupled to the aptamer domain, resulting in a conformational change in the downstream expression domain and determining the signal output. Riboswitch-based biosensing can be extensively applied to the portable real-time detection of food samples. The numerous key features of riboswitch-based biosensing emphasize their sustainability, renewable, and testing, which promises to transform engineering applications in the field of food safety. This review covers recent developments in riboswitch-based biosensors. The brief history, definition, and modular design (regulatory mode, reporter, and expression platform) of riboswitch-based biosensors are explained for better insight into the design and construction. We summarize recent advances in various riboswitch-based biosensors involving theophylline, malachite green, tetracycline, neomycin, fluoride, thrombin, naringenin, ciprofloxacin, and paromomycin, aiming to provide general guidance for the design of riboswitch-based biosensors. Finally, the challenges and prospects are also summarized as a way forward stratagem and signs of progress.

Rebuilding the lid region from conformational and dynamic features to engineering applications of lipase in foods: Current status and future prospects.

The applications of lipases in esterification, amidation, and transesterification have broadened their potential in the production of fine compounds with high cumulative values. Mostly, the catalytic triad of lipases is covered by either one or two mobile peptides called the "lid" that control the substrate channel to the catalytic center. The lid holds unique conformational allostery via interfacial activation to regulate the dynamics and catalytic functions of lipases, thereby highlighting its importance in redesigning these enzymes for industrial applications. The structural characteristic of lipase, the dynamics of lids, and the roles of lid in lipase catalysis were summarized, providing opportunities for rebuilding lid region by biotechniques (e.g., metagenomic technology and protein engineering) and enzyme immobilization. The review focused on the advantages and disadvantages of strategies rebuilding the lid region. The main shortcomings of biotechnologies on lid rebuilding were discussed such as negative effects on lipase (e.g., a decrease of activity). Additionally, the main shortcomings (e.g., enzyme desorption at high temperatre) in immobilization on hydrophobic supports via interfacial action were presented. Solutions to the mentioned problems were proposed by combinations of computational design with biotechnologies, and improvements of lipase immobilization (e.g., immobilization protocols and support design). Finally, the review provides future perspectives about designing hyperfunctional lipases as biocatalysts in the food industry based on lid conformation and dynamics.

Screening and application of a broad-spectrum aptamer for acyclic guanosine analogues.

Acyclic guanosine analogues, a class of widely used antiviral drugs, can cause chronic toxicity and virus resistance. Therefore, it is essential to establish rapid and accurate methods to detect acyclic guanosine analogues. In this study, five acyclic guanosine analogues (acyclovir, famciclovir, ganciclovir, penciclovir, and valaciclovir) were used as positive targets to obtain broad-spectrum aptamers through Capture-SELEX technology. Real-time quantitative PCR (Q-PCR) was used to monitor the aptamer SELEX process. After the sixteen rounds of selection against mixed targets, sequences were obtained by high-throughput sequencing (HTS). Furthermore, a broad-spectrum aptamer, named CIV6, was found as the higher performance aptamer that was suitable for five acyclic guanosine analogues by graphene oxide (GO) polarization and fluorescence assay. Finally, the aptamer CIV6 was used to construct GO fluorescence assay to detect five acyclic guanosine analogues. The limits of detection (LOD) of acyclovir, famciclovir, ganciclovir, penciclovir, and valaciclovir were 0.48 ng·mL-1, 0.53 ng·mL-1, 0.50 ng·mL-1, 0.56 ng·mL-1, and 0.38 ng·mL-1, respectively.

Structure-switching fluorescence aptasensor for sensitive detection of chloramphenicol.

The performance of chloramphenicol aptamer, including binding thermodynamics, structure switching, and binding domain, was investigated by isothermal titration calorimetry, circular dichroism, and molecular docking. Then, a new fluorescence aptasensor was developed with signal amplification mediated by exonuclease I-catalyzed reaction and hybridization chain reaction (HCR) for chloramphenicol detection. In this system, the aptamer-binding domain is blocked by the initiator of HCR, the aptamer undergoes structure switching in the presence of chloramphenicol, and DNA dissociation occurs. The released aptamer is subsequently recognized and cleaved by Exo I to set free chloramphenicol. With the Exo I-assisted chloramphenicol recycling, an increasing number of initiators were exposed from the digestion of the initiator-aptamer complex. Then, the chain-like assembly of FAM labeled H1 and H2 through HCR was triggered by the initiator, generating a long DNA polymer. Under optimum conditions, the aptasensor exhibited a log-linear range from 0.001 to 100 nM of chloramphenicol and a detection limit of 0.3 pM. Additionally, the designed biosensing platform was applied to determine chloramphenicol in milk and lake water with high accuracy. The current approach provides a new avenue to develop sensitive aptasensors with the assistance of binding mechanism between aptamer and target compounds. Graphical abstract.

Aptamer Induced Multicolored AuNCs-WS2 "Turn on" FRET Nano Platform for Dual-Color Simultaneous Detection of AflatoxinB1 and Zearalenone.

Mycotoxins posit serious threats to human and animal health, and numerous efforts have been performed to detect the multiple toxins by a single diagnostic approach. To best of our knowledge, for the first time, we synthesized an aptamer induced "turn on" fluorescence resonance energy transfer (FRET) biosensor using dual-color gold nanoclusters (AuNCs), l-proline, and BSA synthesized AuNCs (Lp-AuNCs and BSA-AuNCs), with WS2 nanosheet for simultaneous recognition of aflatoxinB1 (AFB1) and zearalenone (ZEN) by single excitation. Here, AFB1 aptamer stabilized blue-emitting AuNCs (AFB1-apt-Lp-AuNCs) (at 442 nm) and ZEN aptamer functionalized with red-colored AuNCs (ZEN-apt-BSA-AuNCs) (at 650 nm) were employed as an energy donor and WS2 nanosheet as a fluorescence quencher. With the addition of AFB1 and ZEN, the change in fluorescence intensity (F.I) was recorded at 442 and 650 nm and can be used for simultaneous recognition with a detection limit of 0.34 pg mL-1 (R2 = 0.9931) and 0.53 pg mL-1 (R2 = 0.9934), respectively. Most importantly, the semiquantitative determination of AFB1 and ZEN can also be realized through photovisualization. The current approach paves a new way to develop sensitive, selective, and convenient metal nanocluster-based fluorescent "switch-on" probes with potential applications in multipurpose biosensing.

Simultaneous detection of fumonisin B1 and ochratoxin A using dual-color, time-resolved luminescent nanoparticles (NaYF4: Ce, Tb and NH2-Eu/DPA@SiO2) as labels.

A rapid and highly sensitive time-resolved fluorescence (TRF)-based aptasensor for simultaneous recognition of mycotoxins ochratoxin A (OTA) and fumonisin B1 (FB1) was developed using multi-color, Ln3+-doped time-resolved fluorescence nanoparticles (TRF-NPs) (NaYF4: Ce, Tb and NH2-Eu/DPA@SiO2 NPs) coupled with complementary strand DNA (cDNA) as luminescence probe and aptamers-conjugated amine-functionalized Fe3O4 magnetic nanoparticles (MNPs) act as a capture probe. Under the optimized conditions, the time-resolved fluorescence intensities at 544 and 618 nm corresponded with Tb3+ and Eu3+, respectively, were used to measure FB1 (Y = 19,177.1 + (- 12,054.4)x, R2 = 0.9917) and OTA (Y = 4138.8 + (- 11,182.6)x, R2 = 0.9924), respectively. The limits of detection (LODs) for FB1 and OTA were 0.019 pg mL-1 and 0.015 pg mL-1, respectively, which were much lower than previously described methods for simultaneous recognition of mycotoxins OTA and FB1 while detection range varied from 0.0001-0.5 ng mL-1. This aptasensor was effectively applied to quantity FB1 and OTA in maize samples and results were compared with ELISA method. This is the first reported time-resolved fluorescence (TRF)-based aptasensor to detect two agriculturally important toxins in the maize. The developed aptasensor has potential to be used for detection of toxins in food safety fields. Graphical abstract.

A "turnon" aptasensor for simultaneous and time-resolved fluorometric determination of zearalenone, trichothecenes A and aflatoxin B1 using WS2 as a quencher.

A "turn on" time-resolved fluorometric aptasensor is described for the simultaneous detection of zearalenone (ZEN), trichothecenes A (T-2), and aflatoxin B1 (AFB1). Multicolor-emissive nanoparticles doped with lanthanide ions (Dy3+, Tb3+, Eu3+) were functionalized with respective aptamers and applied as a bioprobe, and tungsten disulfide (WS2) nanosheets are used as a quencher of time-resolved fluorescence. The assay exploits the quenching efficiency of WS2 and the interactions between WS2 and the respective DNA aptamers. The simultaneous recognition of the three mycotoxins can be performed in a single solution. In the absence of targets, WS2 is easily adsorbed by the mixed bioprobes via van der Waals forces between nucleobases and the WS2 basal plane. This brings the bioprobe and WS2 into close proximity and results in quenched fluorescence. In the presence of targets, the fluorescence of the bioprobes is restored because the analytes react with DNA probe and modify their molecular conformation to weaken the interaction between the DNAs and WS2. Under the optimum conditions and at an excitation wavelength of 273 nm, the time-resolved fluorescence intensities (peaking at 488, 544 and 618 nm and corresponding to emissions of Dy3+, Tb3+ and Eu3+) were used to quantify ZEN, T-2 and AFB1, respectively, with detection limits of 0.51, 0.33 and 0.40 pg mL-1 and a linear range from 0.001 to 100 ng mL-1. The three mycotoxins can be detected simultaneously without mutual interference. The assay was applied to the quantification of ZEN, T-2 and AFB1 in (spiked) maize samples. This homogeneous aptamer based assay can be performed within 1 h. Conceivably, it can become an alternative to other heterogeneous methods such as the respective enzyme-linked immunosorbent assays. Graphical abstract Schematic presentation of an aptasensor for simultaneous detection of zearalenone, trichothecenes A and aflatoxin B1 using aptamer modified time-resolved fluorescence nanoparticles as signalling probes and tungsten disulfide as the quencher. This assay shows lower detection limit and requires no washing steps.

Multisystem Inflammatory Syndrome in Neonates Associated with COVID-19 in Neonatal ICU of a Tertiary Care Hospital.

OBJECTIVE: Neonatal multisystem inflammatory syndrome (MIS-N) is a unique disease of neonates described in several case reports from all over the world with a myriad of presentations and the emergence of new cases. STUDY DESIGN: Retrospective case series. Place and Duration of the Study: Department of Paediatrics, Fazaia Medical College, Pakistan Air Force Hospital, Islamabad, Pakistan, from December 2021 to November 2022. METHODOLOGY: The study was conducted on neonates who were managed as MIS-N in the neonatal ICU. Data were collected and analysed on SPSS version 24. RESULTS: Patients in this study ranged from newborns to 13 days of age with a mean age of 3.27 ± 4.29 days and average gestational age of 35.18 ± 3.67 weeks. Among these neonates, 7 (63.6%) had bleeding diathesis, 11 (100%) had seizures, 8 (72.2%) presented with haemodynamic instability and shock, and 7 (63.3%) had signs of heart failure. All neonates (100%) had markedly raised SARS-CoV2 IgG antibodies, CRP, ferritin, D-dimers, interleukin 6, procalcitonin, 10 (90.9%) had hypoalbuminemia, and 7 (63.3%) had deranged coagulation profile. Cardiac involvement was seen in all neonates (100%) with raised proBNP and myocardial dysfunction on echocardiography. Pulmonary hypertension was present in 6 (54.4%) neonates. High mortality was observed at 6 (54.5%) among which 4 (66.6%) were premature neonates. CONCLUSION: MIS-N is a new disease entity which is still under research. There is a high propensity for cardiovascular system involvement and higher mortality among preterm neonates. KEY WORDS: Neonatal multisystem inflammatory syndrome (MIS-N), Multisystem inflammatory syndrome in children (MIS-C), SARS-CoV2 infection, SARS-CoV2 spike protein, SARS-CoV2 IgG antibodies.

Aptamer-modified paper-based analytical devices for the detection of food hazards: Emerging applications and future perspective.

Food analysis plays a critical role in assessing human health risks and monitoring food quality and safety. Currently, there is a pressing need for a reliable, portable, and quick recognition element for point-of-care testing (POCT) to better serve the demands of on-site food analysis. Aptamer-modified paper-based analytical devices (Apt-PADs) have excellent characteristics of high portability, high sensitivity, high specificity, and on-site detection, which have been widely used and concerned in the field of food safety. The article reviews the basic components and working principles of Apt-PADs, and introduces their representative applications detecting food hazards. Finally, the advantages, challenges, and future directions of Apt-PADs-based sensing performance are discussed, to provide new directions and insights for researchers to select appropriate Apt-PADs according to specific applications.

An ultrasensitive dual-mode aptasensor for patulin based on the upconversion particles and G-Quadruplex-hemin DNAzyme.

Patulin (PAT) is a mycotoxin-produced secondary metabolite that can contaminate foods, causing toxic effects on animal and human health. Therefore, for the first time, we have constructed a "turn-on" dual-mode aptamer sensor for PAT using oleic acid-coated upconversion nanomaterials (OA-UCNPs) and G-Quadruplex-hemin DNAzyme (G4-DNAzyme) as fluorescent and colorimetry probes. The sensor employs aptamers binding to PAT as recognition elements for specific molecule detection. Mxene-Au can be used as a biological inducer to assist OA-UCNPs in controlling fluorescence intensity. In addition, colorimetric signal amplification was performed using the trivalent G4-DNAzyme to increase detection sensitivity and reduce false positives. Under optimal conditions, the dual-mode aptasensor has a detection limit of 5.3 pg mL-1 in fluorescence and 2.4 pg mL-1 in colorimetric methods, respectively, with the wider linear range and limit of detection (LOD) of the colorimetric assay. The combination aptasensor can detect PAT with high sensitivity and high specificity and has broad application prospects in the field of food safety detection.

Dual pH- and ATP-Responsive Antibacterial Nanospray: On-Demand Release of Antibacterial Factors, Imaging Monitoring, and Accelerated Healing of Bacteria-Infected Wounds under NIR Activation.

The prevalence of pathogenic bacterial infections with high morbidity and mortality poses a widespread challenge to the healthcare system. Therefore, it is imperative to develop nanoformulations capable of adaptively releasing antimicrobial factors and demonstrating multimodal synergistic antimicrobial activity. Herein, an NIR-activated multifunctional synergistic antimicrobial nanospray MXene/ZIF-90@ICG was prepared by incorporating ZIF-90@ICG nanoparticles onto MXene-NH2 nanosheets. MXene/ZIF-90@ICG can on-demand release the antimicrobial factors MXenes, ICG, and Zn2+ in response to variations in pH and ATP levels within the bacterial infection microenvironment. Under NIR radiation, the combination of MXenes, Zn2+, and ICG generated a significant amount of ROS and elevated heat, thereby enhancing the antimicrobial efficacy of PDT and PTT. Meanwhile, NIR excitation could accelerate the further release of ICG and Zn2+, realizing the multimodal synergistic antibacterial effect of PDT/PTT/Zn2+. Notably, introducing MXenes improved the dispersion of the synthesized antimicrobial nanoparticles in aqueous solution, rendering MXene/ZIF-90@ICG a candidate for application as a nanospray. Importantly, MXene/ZIF-90@ICG demonstrated antimicrobial activity and accelerated wound healing in the constructed in vivo subcutaneous Staphylococcus aureus infection model with NIR activation, maintaining a favorable biosafety level. Therefore, MXene/ZIF-90@ICG holds promise as an innovative nanospray for adaptive multimodal synergistic and efficient antibacterial applications with NIR activation.

Lateral flow assay for simultaneous detection of multiple mycotoxins using nanozyme to amplify signals.

Co-contamination of multiple mycotoxins produces synergistic toxic effects, leading to more serious hazards. Therefore, the simple, rapid and accurate simultaneous detection of multiple mycotoxins is crucial. Herein, a three-channel aptamer-based lateral flow assay (Apt-LFA) was established for the detection of aflatoxin M1 (AFM1), aflatoxin B1 (AFB1) and ochratoxin A (OTA). The multi-channel Apt-LFA utilized gold­iridium nanozyme to catalyze the chromogenic substrate, which effectively achieved signal amplification. Moreover, the positions and lengths of the complementary sequences were screened by changes in fluorescence intensity. After grayscale analysis, the semi-quantitative results showed that the detection limits of AFM1, AFB1 and OTA were 0.39 ng/mL, 0.36 ng/mL and 0.82 ng/mL. The recoveries of the multiplexed competitive sensors in complex matrices of real samples were 93.33%-97.01%, 95.72%-102.67%, and 106.88%-109.33%, respectively. In conclusion, the assembly principle of the three-channel Apt-LFA is simple, which can provide a new idea for the simultaneous detection of small molecule targets.

Fluorescent DNA-Silver nanoclusters in food safety detection: From synthesis to application.

In recent years, the conventional preparation of silver nanoclusters (AgNCs) has attracted much attention due to their ultra-small size, tunable fluorescence, easy-to-engineer, as well as biocompatible material. Moreover, its great affinity towards cytosine bases on single-stranded DNA has led to the construction of biosensors, especially aptamers, for a broad variety of applications in food safety and environmental protection. In past years, numerous researchers paid attention to the construction of AgNCs aptasensor. Therefore, this review will be an effort to summarize the synthetic strategy along with the influences of factors on synthesis, categorize the sensing mechanism of aptamer-functionalized AgNCs biosensors, as well as their specific applications in food safety detection including heavy metal, toxin, and foodborne pathogenic bacteria. Furthermore, a brief conclusion and outlook regarding the prospects and challenges of their applications in food safety were drawn in line with the developments in DNA-AgNCs.

Covalent modification of γ-cyclodextrin with geraniol: An antibacterial agent with good thermal stability, solubility and biocompatibility.

Geraniol (Ger) is an essential oil molecule with excellent biological activity. High hydrophobicity and volatility limit its practical application. Cyclodextrins (CDs) are water-soluble cyclic oligosaccharides with hydrophobic cavities. Physical encapsulation of CDs to improve the solubility and stability of essential oil molecules is not satisfactory. Therefore, this study synthesized the γ-CD derivative (γ-CD-Ger) by grafting Ger onto γ-CD using a bromide-mediated method. Compared to the inclusion complexes (γ-CD/Ger) formed by both, the derivatives exhibit better solubility and thermal stability. The derivative has better antibacterial activity when the ratio of γ-CD to Ger was 1:2. In addition, the derivatives did not exhibit cytotoxic and hemolytic properties. These results indicate that this research provides a water-soluble antibacterial agent with a wide range of promising applications and offers new ideas for the application of alcohol hydrophobic molecules in aqueous systems.

Upconversion fluorescence nanosensor based on enzymatic inhibited and copper-triggered o-phenylenediamine oxidation for the detection of dimethoate pesticides.

Pesticide residues in agricultural products pose a significant threat to human health. Herein, a sensitive fluorescence method employing upconversion nanoparticles was developed for detecting organophosphorus pesticides (OPs) based on the principle of enzyme inhibition and copper-triggered o-phenylenediamine (OPD) oxidation. Copper ions (Cu2+) oxidized the colorless OPD to a yellow 2,3-diaminophenazine (oxOPD). The yellow solution oxOPD quenched the fluorescence of upconversion nanoparticles due to the fluorescence resonance energy transfer. The high affinity of Cu2+ for thiocholine reduced the level of oxOPD, resulting in almost no fluorescence quenching. The addition of dimethoate led to the inhibition of acetylcholinesterase activity and thus prevented the formation of thiocholine. Subsequently, Cu2+ oxidized OPD to form oxOPD, which attenuated the fluorescence signal of the system. The detection system has a good linear range of 0.01 ng/mL to 50 ng/mL with a detection limit of 0.008 ng/mL, providing promising applications for rapid detection of dimethoate.

Corrigendum: Probiotics: mechanism of action, health benefits and their application in food industries.

[This corrects the article DOI: 10.3389/fmicb.2023.1216674.].

Study of binding mechanism of aptamer to kanamycin and the development of fluorescent aptasensor in milk detection.

Aptasensors being versatile sensing platforms presented higher sensitivity toward target detection. However, lacking theoretical basis of recognition between most targets and their corresponding aptamers has impeded their applications. Herein, we conducted a study to explore the binding mechanism of aptamer to kanamycin (Kana) and developed rapid fluorescent aptasensing methods. Based on the fluorescence polarization results, base mutations were performed at different sites of the aptamer. The key binding nucleotides of Kana was identified as T7, T8, C13 and A15 by using isothermal titration calorimetry (ITC). The Kmut3 (2.18 µM) with lower dissociation constants (Kd), one-third of the native aptamer (6.91 µM), was also obtained. In addition, the lower K+ concentration and temperature were found to be conducive to Kana binding. Circular dichroism (CD) results revealed that the binding of Kana can trigger the change of base stacking force and helix force. On the aforementioned basis, a fluorescent sensor was designed with the native aptamer and Kmut3 as recognition elements. The comparison results proved that the Kmut3 presented a 3 times lower limit of detection of 59 nM compared to the native aptamer (148 nM). Notably, this developed aptasensor can be finished in 45 min and was convenient to operate.