Biosensor Method Research Articles

Binding-driven forward tearing protospacer activated CRISPR-Cas12a system and applications for microRNA detection

CRISPR-Cas12a system, characterized by its precise sequence recognition and cleavage activity, has emerged as a powerful and programmable tool for molecular diagnostics. However, current CRISPR-Cas12a-based nucleic acid detection methods, particularly microRNA (miRNA) detection, necessitate additional bio-engineering strategies to exert control over Cas12a activity. Herein, we propose an engineered target-responsive hairpin DNA activator (TRHDA) to mediate forward tearing protospacer activated CRISPR-Cas12a system, which enables direct miRNA detection with high specificity and sensitivity. Target miRNA specifically binding to hairpin DNA can drive forward tearing protospacer in the stem sequence of hairpin structure, facilitating the complementarity between crRNA spacer and protospacer to activate Cas12a. Upon the hairpin DNA as input-responsive activator of Cas12a, a universal biosensing method enables the multiple miRNAs (miR-21, let-7a, miR-30a) detection and also has exceptional capability in identifying single-base mismatches and distinguishing homologous let-7/miR-30 family members. Besides, TRHDA-mediated Cas12a-powered biosensing has realized the evaluation of miR-21 expression levels in diverse cellular contexts by intracellular imaging. Considering the easy programmability of hairpin DNA in responsive region, this strategy could expand for the other target molecules detection (e.g., proteins, micromolecules, peptides, exosomes), which offers significant implications for biomarkers diagnostics utilizing the CRISPR-Cas12a system toolbox.Graphical abstract

Highly Sensitive Electrochemiluminescence Biosensing Method for SARS-CoV-2 N Protein Incorporating the Micelle Probes of Quantum Dots and Dibenzoyl Peroxide Using the Screen-Printed Carbon Electrode Modified with a Carboxyl-Functionalized Graphene.

Obtaining stable electrochemiluminescence (ECL) emissions from a hydrophobic luminophore in aqueous solutions and designing a method without the use of an exogenous coreactant are promising for ECL biosensing. Here, a highly sensitive signal-on ECL immunoassay for the SARS-CoV-2 N protein was developed using micelles as an ECL tag. The micelles were prepared by coencapsulating the luminophore hydrophobic CdSe/ZnS quantum dots and coreactant dibenzoyl peroxide within the hydrophobic core of micelles. The ECL probe was obtained by covalently bonding a SARS-CoV-2 N protein-binding aptamer onto the micelle surface. The construction of the immunosensor was initiated by the immobilization of the anti-SARS-CoV-2 N protein antibody onto the screen-printed carbon electrode (SPCE) with a -COOH-functionalized surface. The surface functionalization of SPCEs was achieved through paste-exfoliated graphene, which was modified with a -COOH group through supramolecular-covalent scaffolds on SPCE. Upon achieving sandwich complexes on the immunosensor, an efficient ECL signal response at -1.4 V versus Ag/AgCl was obtained in phosphate buffer solution. The ECL assay was used for the sensitive determination of SARS-CoV-2 N protein with the linear range from 0.01 to 50 ng mL-1, and the detection limit was 3.0 pg mL-1. The immunosensor showed good reproducibility and stability, and the ECL immunoassay was used to determine the SARS-CoV-2 N protein in serum samples. The proposed approach to obtain micelles is versatile for the preparation of stable ECL luminophores by using hydrophobic materials, and the strategy provides an alternative for ECL bioassays based on the coreactant route.

Just-in-Time Generation of Nanolabels via In Situ Biomineralization of ZIF-8 Enabling Ultrasensitive MicroRNA Detection on Unmodified Electrodes.

Nanolabels can enhance the detection performance of electrochemical biosensing methods, yet their practical application is hindered by complex preparation, batch-to-batch variability, and poor long-term storage stability. Herein, we present a novel electrochemical method for miRNA detection based on the just-in-time generation of zeolitic imidazolate framework-8 (ZIF-8) nanolabels initiated by nucleic acids. In this design, the target miRNA-21 is captured with magnetic beads and polyadenylated by Escherichia coli Poly(A) polymerase (EPP), producing miRNA-21 molecules with poly(A) tails (miR-21-poly(A)). These molecules are then adsorbed onto a bare gold electrode (AuE) surface via adenine-gold affinity interactions, serving as nucleation sites for the rapid in situ formation of ZIF-8 nanoparticles. The ZIF-8 nanoparticles function as signal labels, impeding electron transfer at the electrode interfaces and thereby generating a notable electrochemical signal. The developed method demonstrated exceptional sensitivity, with a detection limit (LOD) as low as 2.3 aM and a linear detection range from 10 aM to 1000 fM. The practical application of the developed method was validated by using it to evaluate miRNA-21 expression levels in various biological samples, including cell lines, tumor tissues, and clinical blood samples from non-small cell lung cancer (NSCLC) patients. This approach simplifies the detection process by eliminating the need for presynthesized nanomaterials and premodified electrodes. Its simplicity and high sensitivity make this method a promising tool for point-of-care testing and a wide range of biomedical research applications.

Micro/Nanorobots for Advanced Light‐Based Biosensing and Imaging

AbstractSensing and imaging of biomolecules are crucial to disease diagnosis, prognosis, and therapy where optical techniques have essential utility. Untethered and remotely controlled micro/nanorobots have shown promising sensing and imaging capabilities, especially in complex biological environments. In this review, how micro/nanorobots are used for optical biosensing and imaging while highlighting the significant developments in the field is discussed. Starting is done by exploring colorimetric biosensing methods enabled by micro/nanorobots. Significant advancements in surface‐enhanced Raman spectroscopy‐integrated micro/nanorobots are reviewed. Further, state‐of‐the‐art optical bio‐imaging applications by micro/nanorobots at in vitro intracellular level are highlighted. Novel in vivo bio‐imaging assisted by optical micro/nanorobot sensors is examined. Furthermore, innovations in micro/nanorobots are assessed where motion augmentation is used as a detection mechanism, with applications in point‐of‐care molecular diagnostics. Finally, the challenges associated with micro/nanorobots‐assisted advanced optical biosensing and imaging while discussing insights about potential research directions for this rapidly progressing field are summarized.

Modeling Mass Transport Dynamics in Deformable Hydrogels during Evaporation.

Hydrogels possess exceptional mechanical properties and biocompatibility, making them widely used in contemporary bioengineering. Specifically, in the development of wearable and implantable health monitoring devices as well as drug delivery systems, hydrogels are utilized to enable precise control over the transport of solutes. Nonetheless, predicting the distribution of substances within hydrogels still poses a significant challenge due to the complex interplay between the movement of water content, migration of solutes, and deformability of the hydrogel polymer network, which presents challenges to theoretical modeling. Our work introduces a numerical model that addresses the movement of water and solute within a flexible hydrogel, accounting for evaporation and/or moisture absorption at the boundary. The model solves for water saturation, solute concentration, and hydrogel deformation iteratively at each time step while computing the boundary movement velocity based on the transport process. By comparing the modeled results of geometry deformation and water and solute distributions during evaporation with our experiments, we demonstrate the accuracy and applicability of our proposed model. This capability to precisely analyze water and solute concentrations in deformable and nonuniform hydrogel environments paves the way for advancements in biosensing and drug delivery methods that rely on elastic porous materials.

Digital One-Step Competitive Detection of a Small Molecule in Synthetic and Environmental Waters.

Optical methods for single-molecule analysis hold the promise of accurate, sensitive, and rapid detection of target molecules. Here, we demonstrate the efficiency of such an approach for the competitive detection of small molecules in water. Our biosensing method is based on a combination of a single-DNA biochip for the parallelization of tethered particle motion real-time measurements with antibodies and modified targets as molecular competitors. The antibodies are coupled to the particles tethered to the surface by a long DNA bearing in its middle the molecular competitor bound to the antibodies. Competitive target binding leads to a detectable conformational change of the DNA tethers from looped to unlooped in proportions related to the target concentration. We thus managed to detect fluorescein, chosen as a model of a target molecule, in freshwater of various qualities, from solutions prepared with ultrapure water to more complex matrices such as river water and wastewater treatment plant effluent samples. Similar dose-response curves were obtained under these various conditions in a wide range of concentrations from nanomolar to micromolar with a limit of detection around 2 nM.

Simultaneous detection of CaMV35S and NOS using fluorescence sensors with dual-emission silver nanoclusters and catalytic hairpin amplification strategy.

Adual-emission fluorescent biosensing method was developed for simultaneous determinationof CaMV35S and NOS in genetically modified(GM) plants. Two designed hairpin DNA (H1, H2) sequences were used as templates to synthesize H1-AgNCs (λex = 570nm, λem = 625nm) and H2-AgNCs (λex = 470nm, λem = 555nm). By using H1-AgNCs and H2-AgNCs as dual-signal tags, combined with signal amplification strategy of magnetic separation to reduce background signal and an enzyme-free catalytic hairpin assembly (CHA) signal amplification strategy, a novel multi-target fluorescent biosensor was fabricated to detect multiple targets based on FRET between signal tags (donors) and magnetic Fe3O4 modified graphene oxide (Fe3O4@GO, acceptors). In the presence of the target NOS and CaMV35S, the hairpin structures of H1 and H2 can be opened respectively, and the exposed sequences will hybridize with the G-rich hairpin sequences HP1 and HP2 respectively, displacing the target sequences to participate in the next round of CHA cycle. Meanwhile, H1-HP1 and H2-HP2 double-stranded DNA sequences (dsDNA) were formed, resulting in the desorption of dsDNA from the surface of Fe3O4@GO due to weak π-π interaction between dsDNA and Fe3O4@GO and leading to the fluorescence recovery of AgNCs. Under optimal conditions, the linear ranges of this fluorescence sensor were 5 ~ 300nmol L-1 for NOS and 5 ~ 200nmol L-1 CaMV35S, and the LODs were 0.14nmol L-1 and 0.18nmol L-1, respectively. In addition, the fluorescence sensor has good selectivity for the detection of NOS and CaMV35S in GM soybean samples, showing the potential applications in GM screening.

A Bis(Acridino)-Crown Ether for Recognizing Oligoamines in Spermine Biosynthesis.

Oligoamines in cellular metabolism carry extremely diverse biological functions (i.e., regulating Ca2+-influx, neuronal nitric oxide synthase, membrane potential, Na+, K+-ATPase activity in synaptosomes, etc.). Furthermore, they also act as longevity agents and have a determinative role in autophagy, cell growth, proliferation, and death, while oligoamines dysregulation is a key in a variety of cancers. However, many of their mechanisms of actions have just begun to be understood. In addition to the numerous biosensing methods, only a very few simple small molecule-based tests are available for their selective but reversible tracking or fluorescent labeling. Motivated by this, we present herein a new fluorescent bis(acridino)-crown ether as a sensor molecule for biogenic oligoamines. The sensor molecule can selectively distinguish oligoamines from aliphatic mono- and diamino-analogues, while showing a reversible 1:2 (host:guest) complexation with a stepwise binding process accompanied by a turn-on fluorescence response. Both computational simulations on molecular docking and regression methods on titration experiments were carried out to reveal the oligoamine-recognition properties of the sensor molecule. The new fluorescent chemosensor molecule has a high potential for molecular-level functional studies on the oligoamine systems in cell processes (cellular uptake, transport, progression in cancers, etc.).

Rapid detection of African swine fever virus via SERS probe-modified sandwich hybridization assay.

Rapid and reliable detection method for African swine fever virus (ASFV) is proposed by surface-enhanced Raman spectroscopy (SERS). The ASFV target DNA can be specifically captured by sandwich hybridization between nanomagnetic beads and a SERS probe. Experimental results show that the significant Raman signal of the SERS probe with gold nanoparticles and a molecular reporter DTNB (5,5'-dimercapto-bis (2-nitrobenzoic acid)) can be adopted for detecting the hybridization chain reaction of ASFV DNA. The advantage of the SERS sandwich hybridization assay is the large response range from the single molecule level to 108 copies per mL, which not only can overcome the tedious timerequired for theamplification reaction but also provides a comparative method to polymerase chain reaction. Furthermore, real samples of African swine fever virus were detected from different subjects of swine fever virus including porcine reproductive respiratory syndrome virus and Japanese encephalitis virus. The proposed biosensor method can rapidly detect ASFV correctly within 15min as a simple, convenient, low-cost detection approach. The biosensor can be used as a platform for the determination in biological, food, and environmental analytical fields.

Detection of colorectal cancer associated circular RNAs hsa_circ_0031263, hsa_circ_0072715, and hsa_circ_0136666 in plasma with nanowire chips

Rationale: Colorectal cancer (CRC) is one of the most prevalent oncological diseases with high mortality. Invasive optical (endoscopic) colono- scopy has been recognized as a golden standard for the CRC diagnostics. A promising area is the development of non-invasive tools for CRC diagnosis with circular RNA (circRNA). One of the most sensitive non-invasive tools for detection of cancer RNA markers is considered to be the biosensor methods with the use of nanowire chips with oDNA probes (fragments of DNA oligonucleotides) immobilized on their surface. It has been previously shown that circRNA hsa_circ_0136666_CBC1, hsa_circ_0031263_CBC1, and hsa_circ_0072715_CBC1 are associated with CRC. Aim: To determine the lower limit of concentration sensitivity of detection of CRC-associated circRNA with nanowire chips with immobilized oDNA probes, to demonstrate the usability of these chips for non-invasive detection of circRNA in plasma in the CRC diagnostics, and to establish the potential for the use of nanowire chips for the early CRC diagnosis. Methods: To ensure biospecific binding of the circRNA hsa_circ_0136666_CBC1, hsa_circ_0031263_CBC1, and hsa_circ_0072715_CBC1 (the CRC markers), oDNA probes with the nucleotide sequences complementary to the target circRNA have been immobilized on the nanowire surface. At the study step 1, we detected the lower concentration limit for detection of the target molecules with the use of their analogues, i.e. synthetic model oDNA with the nucleotide sequences complementary to oDNA probes. At the study step 2, we used the nanowire chips with immobilized oDNA probes to detect the circRNA in plasma of the patients with confirmed CRC. Plasma samples from non-cancer patients were used as controls. Results: The lower concentration limit for the detection of DNA analogues of the circRNA hsa_circ_0136666_CBC1, hsa_circ_0031263_CBC1, and hsa_circ_0072715_CBC1 with nanowire chips with oDNA probes was 10-16 М. The analysis of total RNA isolated from plasma of the CRC patients showed a significant increase in the signal from the sensory elements of the nanowire chip. The analysis of plasma samples from the non-cancer patients, the nanowire signal changes were non-significant indicating the absence of detectable concentrations of the circRNA in plasma of the non-cancer patients. Conclusion: We have identified the minimal detectable concentration of the circRNA hsa_circ_0136666_CBC1, hsa_circ_0031263_CBC1, and hsa_circ_0072715_CBC1, associated to the development of CRC, with nanowire chips with immobilized oDNA probes: it was 10-16 М. The experiment showed the usability of such nanowire chips for non-invasive detection of the given circRNA markers in total RNA samples isolated from plasma of CRC patients.

CRISPR/Cas12a cleavage triggered nanoflower for fluorescence-free and target amplification-free biosensing of ctDNA in the terahertz frequencies.

The detection of tumor biomarkers in liquid biopsies requires high sensitivity and low-cost biosensing strategies. However, few traditional techniques can satisfy the requirements of target amplification-free and fluorescence-free at the same time. In this study, we have proposed a novel strategy for ctDNA detection with the combination of terahertz spectroscopy and the CRISPR/Cas12 system. The CRISPR/Cas12a system is activated by the target ctDNA, resulting in a series of reactions leading to the formation of an Au-Fe complex. This complex is easily extracted with magnets and when dropped onto the terahertz metamaterial sensor, it can enhance the frequency shift, providing sensitive and selective sensing of the target ctDNA. Results show that the proposed terahertz biosensor exhibits a relatively low detection limit of 0.8 fM and a good selectivity over interference species. This detection limit is improved by three orders of magnitude compared with traditional biosensing methods using terahertz waves. Furthermore, a ctDNA concentration of 100 fM has been successfully detected in bovine serum (corresponding to 50 fM in the final reaction system) without amplification.

Heavily Labeled Signal Probe for Electrogenerated Chemiluminescence Peptide-Based Biosensing of Matrix Metalloproteinase 2.

The field of electrogenerated chemiluminescence (ECL) biosensing has witnessed remarkable growth, emphasizing the need for precise detection of biomarkers. The synthesis approach of peptide-based signal probe with high recognition ability and high ECL efficiency is a significant issue in the ECL biosensing. Here, a heavily labeled signal probe was synthesized for ECL peptide-based biosensing tactic by using a new aldehyde bearing cyclometalated Ir(III) complex ([Ir(bt)2(bpy-CHO)PF6 (bt=2-phenylbenzothiazole, bpy-CHO=4'-methyl-[2,2'-bipyridine]-4-carbaldehyde, denoted as Ir1) as ECL signal reagent and streptavidin (SA) as carrier protein. One ECL peptide-based biosensing method was exemplified for the detection of matrix metalloproteinase 2 (MMP-2) by using Ir1 labeled SA (SA-Ir1) as heavily labeled signal probe and biotinylated peptide as molecular recognition substrate. MMP-2 was sensitively detected in the range from 5 to 100 ng/mL with a detection limit of 1.5 ng/mL. Importantly, two detection modes differing in the order of cleavage recognition by MMP-2 and signal transduction with SA-Ir1 were compared for the first time. First cleavage and second signal transduction were proposed to be beneficial to sensitive detection of target, which provides some ideas for biomarker diagnostics in disease screening at an early stage.

A turn-on anthraquinone-derived colorimetric and fluorometric dual-mode probe for highly selective Hg2+ determination and bioimaging in living organisms

Mercury ion (Hg2+) is considered a harmful neurotoxin, and real-time monitoring of Hg2+ concentrations in environmental and biological samples is critical. Fluorescent probes are a rapidly emerging visualization tool owing to their simple design and good selectivity. Herein, a novel fluorescence (FL) probe 2-(4-((6-((quinolin-8-yloxy)methyl)pyridin-2-yl)methyl)piperazin-1-yl)anthracene-9,10-dione (QPPA) is designed using piperazine as a linker between the anthraquinone group, which serves as a fluorophore, and N4O as the Hg2+ ligand. The probe exhibits FL “turn-on” sensing of Hg2+ because the complex inhibits the photo-induced electron transfer (PET) process. Moreover, QPPA can overcome the invasion by other possible cations, resulting in a clear color change from orange to colorless with the addition Hg2+. The chelation of QPPA with Hg2+ in a 1:1 ratio. Subsequently, the theoretically determined binding sites of the ligand to Hg2+ are validated through 1H NMR titration. The in situQPPA–Hg2+ complex can be subjected to Hg2+ extraction following the introduction of S2− owing to its robust binding capacity. The exceptional limit of detection values for Hg2+ and S2− are obtained as 63.0 and 79.1 nM (S/N = 3), respectively. Moreover, QPPA can display bright red FL in the presence of Hg2+ in different biological specimens such as HeLa cells, zebrafish, onion root tip tissues, and water flea Daphnia carinata, further providing an effective strategy for environmental monitoring and bioimaging of Hg2+ in living organisms.

Research Progress of Biosensor Based on Organic Photoelectrochemical Transistor.

In order to solve the food safety problem better, it is very important to develop a rapid and sensitive technology for detecting food contamination residues. Organic photoelectrochemical transistor (OPECT) biosensor rely on the photovoltage generated by a semiconductor upon excitation by light to regulate the conductivity of the polymer channels and realize biosensor analysis under zero gate bias. This technology integrates the excellent characteristics of photoelectrochemical (PEC) bioanalysis and the high sensitivity and inherent amplification ability of organic electrochemical transistor (OECT). Based on this, OPECT biosensor detection has been proven to be superior to traditional biosensor detection methods. In this review, we summarize the research status of OPECT biosensor in disease markers and food residue analysis, the basic principle, classification, and biosensing mechanism of OPECT biosensor analysis are briefly introduced, and the recent applications of biosensor analysis are discussed according to the signal strategy. We mainly introduced the OPECT biosensor analysis methods applied in different fields, including the detection of disease markers and food hazard residues such as prostate-specific antigen, heart-type fatty acid binding protein, T-2 toxin detection in milk samples, fat mass and objectivity related protein, ciprofloxacin in milk. The OPECT biosensor provides considerable development potential for the construction of safety analysis and detection platforms in many fields, such as agriculture and food, and hopes to provide some reference for the future development of biosensing analysis methods with higher selectivity, faster analysis speed and higher sensitivity.

Neural Network Enables High Accuracy for Hepatitis B Surface Antigen Detection with a Plasmonic Platform.

The detection of hepatitis B surface antigen (HBsAg) is critical in diagnosing hepatitis B virus (HBV) infection. However, existing clinical detection technologies inevitably cause certain inaccuracies, leading to delayed or unwarranted treatment. Here, we introduce a label-free plasmonic biosensing method based on the thickness-sensitive plasmonic coupling, combined with supervised deep learning (DL) using neural networks. The strategy of utilizing neural networks to process output data can reduce the limit of detection (LOD) of the sensor and significantly improve the accuracy (from 93.1%-97.4% to 99%-99.6%). Compared with widely used emerging clinical technologies, our platform achieves accurate decisions with higher sensitivity in a short assay time (∼30 min). The integration of DL models considerably simplifies the readout procedure, resulting in a substantial decrease in processing time. Our findings offer a promising avenue for developing high-precision molecular detection tools for point-of-care (POC) applications.

Enhancing Early Detection of Sepsis in Neonates through Multimodal Biosignal Integration: A Study of Pulse Oximetry, Near-Infrared Spectroscopy (NIRS), and Skin Temperature Monitoring.

Sepsis continues to be challenging to diagnose due to its non-specific clinical signs and symptoms, emphasizing the importance of early detection. Our study aimed to enhance the accuracy of sepsis diagnosis by integrating multimodal monitoring technologies with conventional diagnostic methods. The research included a total of 121 newborns, with 39 cases of late-onset sepsis, 35 cases of early-onset sepsis, and 47 control subjects. Continuous monitoring of biosignals, including pulse oximetry (PO), near-infrared spectroscopy (NIRS), and skin temperature (ST), was conducted. An algorithm was then developed in Python to identify early signs of sepsis. The model demonstrated the capability to detect sepsis 6 to 48 h in advance with an accuracy rate of 87.67 ± 7.42%. Sensitivity and specificity were recorded at 76% and 90%, respectively, with NIRS and ST having the most significant impact on predictive accuracy. Despite the promising results, limitations such as sample size, data variability, and potential biases were noted. These findings highlight the critical role of non-invasive biosensing methods in conjunction with conventional biomarkers and cultures, offering a strong foundation for early sepsis detection and improved neonatal care. Further research should be conducted to validate these results across different clinical settings.

Development of rapid and efficient enterovirus typing and classification platform for real-time diagnostics

Enteroviruses are highly prevalent among children and adults worldwide. Accurate detection and genotyping of enteroviruses can be achieved using consensus-degenerate hybrid oligonucleotide primer polymerase chain reaction (PCR)-based methods. Nevertheless, the popularized use of such methods for enterovirus detection and genotyping is limited by their relatively long processing time (>2 days), high processing cost, and the requirement for specialized and expensive instrumentation. Here, we report a relatively simple asymmetric PCR-electrochemical biosensing method that can detect enteroviruses and accurately characterize them into four distinct genotypic subgroups in less than 2 h. Specifically, genes of interest in the enteroviruses were amplified via asymmetric PCR (asy-PCR) using the designed PCR primers. The asy-PCR products were hybridized with enterovirus genotype-specific single-stranded DNA primer probes. Changes in response to electrochemical impedance signals can rapidly clarify the genotypes of enteroviruses. We observed that this method could not only effectively amplify the target gene within minimal PCR-amplified cycles of consensus-degenerate hybrid oligonucleotide (COHOP) products from the specimen but also allowed the rapid differentiation of the four subgroups of enteroviruses in 2 h. In summary, we developed an efficient and rapid electrochemical biosensing platform for enterovirus subtyping diagnostics that can be applied to other emergent infectious diseases.

Сучасні підходи до індикації Bacillus anthracis у навколишньому середовищі

Bacillus anthracis — the causative agent of anthrax, which can be used for bioterrorism purposes. An effective biosecurity strategy involves the improvement diagnostics tests for increase its sensitivity and specificity, and the tests for the rapid detection of pathogen. The literature review presents the results of recent methods of detecting Bacillus anthracis spores/cells in environmental objects with greatest epidemiological significance — in air and soil. Conventional microbiological methods are the gold standard for bacterial identification, but their limitation is necessity to cultivate and work with a live particularly dangerous pathogen — B. anthracis, which is associated with the risk of laboratory infection. Now, a number of new methods have been developed for the indication B. anthracis, which allow speed up the process of their detection and are much safer from the point of view of biosecurity. First of all, these are methods based on DNA amplification. Sequencing the DNA of the pathogen adds specificity to the method, and also identifies rare or new variants of B. anthracis. A rapid method for the identification of B. anthracis DNA, which takes less than an hour, has been developed. Also, immunological methods are used to determine the pathogen’s antigen. It should be noted that the specific identification of B. anthracis remains difficult due to its phenotypic and genotypic similarity to other Bacillus species. To solve this problem, a phage typing method was proposed, which has recently been improved with the help of biotechnological methods (construction of the B. anthracis phage) with the detection of its bioluminescence. A promising technology for fast, highly sensitive and specific detection and identification of B. anthracis is biosensor methods using portable devices for their implementation. Key words: anthrax, Bacillus аnthracis, environment, diagnostic tests.

Оптимизация технологии фагового дисплея для получения антител, специфичных к тетрациклину

Large-scale production and use of antimicrobials in human, veterinary and agricultural applications has led to antibiotic contamination of water resources. Therefore, it is relevant to develop methods for monitoring the content of antibiotics, especially in water resources. Biosensor methods are successful for the analysis of antibiotics. One of the main elements of a biosensor system is the selection of a recognition element. An alternative tool for selecting a sensitive element (recognition element) is phage display of antibodies, which makes it possible to obtain antibodies to low molecular weight antigens. The purpose of the work was to conduct research to develop and optimize the methodology for obtaining antibodies specific to tetracycline by using phage display technology, and to evaluate the possibility of their use for the indication of tetracycline. The work included studies of optimization of conditions for the production of phage antibodies specific to tetracycline, as well as the preservation of their activity during storage. The promise of using phage display technology for the production of anti-tetracycline antibodies has been shown. Using the dot immunoassay method, the possibility of using the obtained phage antibodies for tetracycline detection was shown.

A biosensor method based on surfactant-mediated surface droplet evaporation for the detection of Aflatoxin B1

Aflatoxin B1 (AFB1) is a common mycotoxin that is of significant global concern due to its impact on food safety. Herein, we innovatively develop a sensing platform to detect AFB1 based on evaporation of surfactant solutions on the hydrophobic surface, resulting in dried patterns with varied sizes. The surfactant CTAB solution produces a relatively large dried pattern due to the surface wetting. However, the reduction in the dried pattern size is found when the mixture of CTAB and AFB1 aptamer is tested, because the formation of CTAB/aptamer complex. Moreover, the dried pattern size of the mixture of CTAB, aptamer, and AFB1 increases due to the specific binding of AFB1 to its aptamer. Using this innovative strategy, the AFB1 detection can be fulfilled with a detection limit of 0.77 pg/mL. As a simple, convenient, inexpensive, and label-free method, the surfactant-mediated surface droplet evaporation-based biosensor is very promising for various potential applications.