Signal amplification strategy of DNA self-assembled biosensor and typical applications in pathogenic microorganism detection.

Biosensors have emerged as ideal analytical devices for various bio-applications owing to their low cost, convenience, and portability, which offer great potential for improving global healthcare. DNA self-assembly techniques have been enriched with the development of innovative amplification strategies, such as dispersion-to-localization of catalytic hairpin assembly, and dumbbell hybridization chain reaction, which hold great significance for building biosensors capable of realizing sensitive, rapid and multiplexed detection of pathogenic microorganisms. Here, focusing primarily on the signal amplification strategies based on DNA self-assembly, we concisely summarized the strengths and weaknesses of diverse isothermal nucleic acid amplification techniques. Subsequently, both single-layer and cascade amplification strategies based on traditional catalytic hairpin assembly and hybridization chain reaction were critically explored. Furthermore, a comprehensive overview of the recent advances in DNA self-assembled biosensors for the detection of pathogenic microorganisms is presented to summarize methods for biorecognition and signal amplification. Finally, a brief discussion is provided about the current challenges and future directions of DNA self-assembled biosensors.

Platelet-Based Nanoparticles with Stimuli-Responsive for Anti-Tumor Therapy.

In addition to hemostasis and coagulation, years of studies have proved that platelets are involved in the whole process of tumor progression, including tumor invasion, intravasation, extravasation, and so on. It means that this property of platelets can be used in anti-tumor therapy. However, traditional platelet-based antitumor drugs often cause autologous platelet damage due to lack of targeting, resulting in serious side effects. Therefore, the researchers designed a variety of anti-tumor drug delivery systems based on platelets by targeting platelets or platelet membrane coating. The drug delivery systems have special response modes, which is crucial in the design of nanoparticles. These modes enhance the targeting and improve the anti-tumor effect. Here, we present a review of recent discoveries in the field of the crosstalk between platelets and tumors and the progress of platelet-based anti-tumor nanoparticles.

A dual fragment triggered DNA ladder nanostructure based on logic gate and dispersion-to-localization catalytic hairpin assembly for efficient fluorescence assay of SARS-CoV-2 and H1N1.

DNA nanotechnology has been widely utilized in the construction of various functional nanostructures. However, most DNA nanostructures have the shortcomings of low response rate and serious background leakage. Herein, we proposed the conception of AND logic gate cascaded dispersion-to-localization catalytic hairpin assembly (AND gate-DLCHA) for the fabrication of novel DNA ladder nanostructures. In our design, the entropy-driven AND logic gate can precisely recognize two fragments of the target nucleic acid sequences. After AND logic gate activation by target nucleic acids, dispersion-to-localization catalytic hairpin assembly was initiated. Consequently, tremendous DNA ladder nanostructures were generated and the response signal was rapidly enhanced, which can be used for rapid and amplied detection of nucleic acids. Taking advantage of the sensitivity and specificity of AND gate-DLCHA strategy, the fluorescence sensors were established and successfully applied in ultrasensitive assay of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (H1N1) within 45 min with the limit of detection (LOD) as low as 66 copies mL-1 (SARS-CoV-2) and 33 copies mL-1 (H1N1), which showed perspectives in pathogen identification and biomedical application. The high selectivity and reliability of established sensors was attributed to the dual-fragment analysis. Meanwhile, the sensors possessed minimal leakage and greatly enhanced signal to background (S/B) ratio owing to substrate transduction from dispersion into colocalization. This rationally developed logic gate cascaded dispersion-to-localization catalytic hairpin assembly strategy presented a new approach for the development of DNA nanostructures.

Isothermal amplification based on specific signal extraction and output for fluorescence and colorimetric detection of nucleic acids.

Due to the complexity of compositions and low abundance of target in clinical sample, nucleic acids detection often suffers from false positives caused by nonspecific amplification. In in vitro diagnosis (IVD), PCR usually employ TaqMan probe to report specific signals and block false positive signals. However, nucleic acid isothermal amplifications, such as loop-mediated isothermal amplification (LAMP), lack of mature specific signal output mechanism, which prevents them from being used for IVD and point-of-care testing (POCT). In this work, we constructed a specific signal extract-to-output isothermal detection system (SSEI). SSEI contains a well-designed DNA probe for specific signal extraction and output in LAMP. This probe is a double-stranded DNA with an overhang sequence and named as extract-to-output probe (ETO probe). ETO probe can recognize the target-specific intermediate products in LAMP and release another signal-output probe (OP) to report the target-specific signals. With these unique properties, SSEI can detect as low as 10 copies of target DNA per reaction either by fluorescence detector or naked eyes. Moreover, due to the excellent performance against background nucleic acids interference, this biosensing platform had been successfully used for hepatitis B virus (HBV) clinical samples detection.

Sensitive fluorescence biosensor for SARS-CoV-2 nucleocapsid protein detection in cold-chain food products based on DNA circuit and g-CNQDs@Zn-MOF.

SARS-CoV-2 isolation from cold-chain food products confirms the possibility of outbreaks through cold-chain food products. RNA extraction combined with RT-PCR is the primary method currently utilized for the detection of SARS-CoV-2. However, the requirement of hours of analytical time and the high price of RT-PCR hinder its worldwide implementation in food supervision. Here, we report a fluorescence biosensor for detection of SARS-CoV-2 N protein. The fluorescence biosensor was fabricated by aptamer-based conformational entropy-driven circuit where molecular beacon strands were labeled with graphitic carbon nitrides quantum dots@Zn-metal-organic framework (g-CNQDs@Zn-MOF) and Dabcyl. The detection of the N protein was achieved via swabbing followed by competitive assay using a fixed amount of N-48 aptamers in the analytical system. A fluorescence emission spectrum was employed for the detection. The detection limit of our fluorescence biosensor was 1.0 pg/mL for SARS-CoV-2 N protein, indicating very excellent sensitivity. The fluorescence biosensor did not exhibit significant cross-reactivity with other N proteins. Finally, the biosensor was successfully applied for the detection of SARS-CoV-2 N protein in actual cold-chain food products showing same excellent accuracy as RT-PCR method. Thus, our fluorescence biosensor is a promising analytical tool for rapid and sensitive detection of SARS-CoV-2 N protein.

Plasma colorimetric aptasensor for the detection of chloramphenicol in honey based on cage Au@AuNPs and cascade hybridization chain reaction.

A plasma colorimetric aptasensor was developed for rapid determination of chloramphenicol (CAP) in honey on site. Herein, cage gold shell@core nanoparticles (Au@AuNPs) were synthesized to enhance signal response and broaden the linear range. In addition, aptamer-based cascade hybridization chain reaction (cHCR), consisting of HP1, HP2, HP3, and HP4, was also designed for signal amplification and specific analysis. In this assay, HP1 and HP4 were immobilized on the surface of cage Au@AuNPs. In the presence of CAP, cHCR was triggered, and frond-like DNA products were formed, which made the distance among the cage Au@AuNPs closer and the system color changed from red to deep purple. Qualitative and quantitative analysis were carried out according to color changes and UV-Vis spectra. Under the optimized conditions, the wavelength of UV-Vis absorption peak exhibited a good linear relationship with CAP concentration in the range of 5.0 to 500 nmol/L with the detection limit of 1.2 nmol/L (S/N = 3). This aptasensor also showed good specificity for CAP detection compared with other antibiotics similar to the target analyte. Furthermore, the colorimetric aptasensor was successfully applied to the detection of CAP in honey with recoveries of 88.0-107.6%. This cHCR-based aptasensing for CAP possesses high sensitivity, good selectivity, low cost and excellent stability, and could be extended to detect a wide variety of other small molecular analytes, nucleic acids or proteins. Therefore, the versatile method might become a potential alternative tool in food analysis and environmental monitoring.

Recent advances in biosensors for antibiotic detection: Selectivity and signal amplification with nanomaterials.

Antibiotics are widely used in the prevention and treatment of infectious diseases in animals due to its bactericidal or bacteriostatic action. Residual antibiotics and their metabolites pose great threats to human and animal health, such as potential carcinogenic and mutagenic effects, and bacterial resistances. Therefore, it is necessary and urgent to accurately monitor trace amounts of antibiotics in food samples. Up to now, many analytical methods have been reported for the determination of antibiotics. Biosensors with the advantages of high sensitivity, rapid response, easy miniaturization, and low price have been widely applied to the detection of antibiotics residues in past decades. This review offered an in-depth evaluation of recognition elements for antibiotic residues in diverse food matrices. In addition, it presented a systematical and critical review on signal amplification via various materials, focusing on recently developed nanomaterials. Finally, the review provided an outlook on the future concepts to help upgrade the sensing techniques for antibiotics in food.

Speciation analysis of arsenic in edible mushrooms by high-performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry.

Some varieties of edible mushrooms can accumulate high contents of arsenic, which is a public health concern. In this study, we developed a high-performance liquid chromatography method linked to inductively coupled plasma triple quadrupole mass spectrometry (HPLC-ICP-MS/MS) for sensitive and accurate determination of arsenite, arsenate, monomethylarsonic acid, dimethylarsinic acid, arsenocholine and arsenobetaine in edible mushrooms. The six arsenic species were extracted ultrasonically from mushrooms using ultrapure water at 60 °C as the solvent, separated on a PRP-X100 anion exchange column (4.1 × 250 mm, 10 µm), with 20 mmol/L NH4HCO3 and 50 mmol/L (NH4)2CO3 as the mobile phase, and quantified using ICP-MS/MS in the oxygen reaction mode. The linear range of the method was 0.5 µg/L-100 µg/L with detection and quantification limits of 2.5 µg/kg-10 µg/kg (S/N = 3), and 8 µg/kg-33 µg/kg (S/N = 10), respectively. This method was applied successfully to the detection and speciation of arsenic in eight varieties (266 samples) of mushrooms. Our results indicated that most wild edible mushrooms contained organic arsenic, mainly arsenobetaine and arsenocholine. However, the inorganic arsenic content of Armillariella tabescens (3.63 mg/kg) and parts of the cultivated Agaricus blazei murrill (up to 4.50 mg/kg) were relatively high, which is potential risk to the health of consumers.

Speciation analysis of mercury in wild edible mushrooms by high-performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry.

Wild edible mushrooms can accumulate significantly elevated levels of mercury from the surrounding environment, which could be harmful to consumers' health. Speciation analysis of mercury in wild edible mushrooms aids in understanding the human exposure to these toxic compounds. In this study, we developed a high-performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (HPLC-ICP-MS) method for the simultaneous determination of inorganic mercury (Hg(II)), methylmercury (MeHg), ethylmercury (EtHg), and phenylmercury (PhHg) in wild edible mushrooms. A rapid separation of four target mercury species was achieved within 11 min by a C8 column without utilizing high proportion of organic phase in HPLC. The parameters affecting the extraction efficiency of mercury in samples have been investigated. The proposed method showed good linearity within 0-50 µg/L with the detection and quantification limits of 0.6-4.5 µg/kg (S/N = 3), and 2.0-15 µg/kg (S/N = 10), respectively. This proposed method was successfully applied to the mercury speciation analysis in 7 varieties (95 samples) of wild edible mushrooms. The results indicated that in most mushroom samples, mercury mainly occurred as inorganic mercury. But there were two Tricholoma matsutakes, one contained 0.14 mg/kg of methylmercury, another contained 1.05 mg/kg of phenylmercury, which were higher than the maximum allowable content of total mercury in edible mushrooms in China. Graphical abstract.

Occurrence, toxicity, and speciation analysis of arsenic in edible mushrooms.

Owing to the strong concentration and biotransformation of arsenic, the influence of some edible mushrooms on human health has attracted widespread attention. The toxicity of arsenic greatly depends on its species, so the speciation analysis of arsenic is of critical importance. The aim of the present review is to highlight recent advances in arsenic speciation analysis in edible mushrooms. We summarized the contents and distribution of arsenic species in some edible mushrooms, the methods of sample preparation, and the techniques for their identification and quantification. Stability of the arsenic species during sample pretreatment and storage is also briefly discussed.

Signal amplification strategies for DNA-based surface plasmon resonance biosensors.

DNA has well-defined ability to recognize a wide variety of targets, such as small biological molecules, proteins, inorganic ions and small organic molecules. As molecular recognition elements, DNA can be used to build simple, rapid and sensitive biosensors for detection of these targets. DNA-based SPR sensors are considered to be a real-time and label-free tool. We present a systematical and critical review on DNA-based SPR biosensors and their signal amplification via various strategies, focusing on recent advances in nanomaterials, novel DNA amplifications, redox reactions on surface, enzyme amplifications, as well as promising multiplex amplification strategies.

Fiber optic surface plasmon resonance sensor for detection of E. coli O157:H7 based on antimicrobial peptides and AgNPs-rGO.

A fiber optic surface plasmon resonance (FOSPR) sensor was developed for detection of Escherichia coli O157:H7 (E. coli O157:H7) in water and juice, based on antimicrobial peptides (AMP), Magainin I, as recognition elements and silver nanoparticles-reduced graphene oxide (AgNPs-rGO) nanocomposites assisted signal amplification. The uniform AgNPs-rGO was fixed on the surface of optical fiber and covered with gold film. Not only was the SPR response greatly enhanced, but also the AgNPs was prevented from being oxidized. The FOSPR showed a sensitivity of about 1.5 times higher than that fabricated only with gold film. In the assay, Magainin I, immobilized on the surface of gold film, could specifically capture E. coli O157:H7, resulting in the wavelength shift of the SPR absorption peak. Under the optimized conditions, the SPR resonance wavelength exhibited a good linear relationship with natural logarithm of the target bacteria concentration in the range of 1.0 × 103 to 5.0 × 107 cfu/mL with the detection limit of 5.0 × 102 cfu/mL (S/N = 3). The FOSPR sensor showed good specificity for E. coli O157:H7 detection compared to other bacteria similar to the target bacterial species. Furthermore, the FOSPR sensor was successfully applied to the detection of E. coli O157:H7 in water, fruit and vegetable juice with the satisfactory recoveries of 88-110%. This assay for E. coli O157:H7 detection possesses high sensitivity, good selectivity, reproducibility and stability. In addition, the AMP based SPR biosensing methodology could be extended to detect a wide variety of foodborne pathogens. Therefore, the versatile method might become a potential alternative tool in food analysis and early clinical diagnosis.

Fluorescent aptasensor for detection of four tetracycline veterinary drugs in milk based on catalytic hairpin assembly reaction and displacement of G-quadruplex.

Based on a novel signal amplification strategy by catalytic hairpin assembly and displacement of G-quadruplex DNA, an enzyme-free, non-label fluorescent aptasensing approach was established for sensitive detection of four tetracycline veterinary drugs in milk. The network consisted of a pair of partially complementary DNA hairpins (HP1 and HP2). The DNA aptamer of four tetracycline veterinary drugs was located at the sticky end of the HP1. The ring region of HP1 rich in G and C could form a stable G-quadruplex structure, which could emit specific fluorescence signal after binding with the fluorescent dye and N-methylmesoporphyrin IX (NMM). When presented in the system, the target analytes would be repeatedly used to trigger a recycling procedure between the hairpins, generating numerous HP1-HP2 duplex complexes and displacing G-quadruplex DNA. Thus, the sensitive detection of target analytes was achieved in a wide linear range (0-1000 µg/L) with the detection limit of 4.6 µg/L. Moreover, this proposed method showed high discrimination efficiency towards target analytes against other common mismatched veterinary drugs, and could be successfully applied to the analysis of milk samples. Graphical abstract Schematic of target analyte detection based on catalytic hairpin assembly reaction and displacement of G-quadruplex.

[Technology and MethodsSimultaneous Determination of 9 Natural Ingredientsin Functional Food for Anti-hangover and Hepatoprotection by High Performance Liquid Chromatography].

OBJECTIVES: To develop a rapid method of high performance liquid chromatography coupled with variable wave length UV detection for simultaneous determination of 9 natural functional ingredients including puerarin, silymarin, quercetin hydrate, schisandrol A, curcumin, tanshinoneI, tanshinoneIIA, cryptotanshinone, and dihydrotanshinoneIin functional food for anti-hangover and hepatoprotection. METHODS: The samples were ultrasonically extracted with 90 % ethanol (V/V) and centrifuged at 10 000 r/min for 10 min prior to HPLC analysis. The nine target analytes were separated on a C18 column with gradient elution using methanol and water (The pH value was adjusted to 2.5 using H3PO4) as the mobile phase. Qualitative analysis was carried out using retention times of the chromatographic peaks, while the external standard curves were established for quantification. RESULTS: Under the optimal analytical conditions, the peak area of each analyte and its concentration had a good correlation within the linear range ( r≥0.998). The limits of detection and quantification of the method were 0.38-0.73 mg/kg (S/N=3) and 1.27-2.43 mg/kg (S/N=10), respectively. The spiked recoveries of the analytes were 88.9%-103.2%, and the relative standard deviations were 1.3%-3.7%. CONCLUSIONS: The method for the determination of 9 functional ingredients in functional food for anti-hangover and hepatoprotection was proposed for the first time in this study. The results showed that it could meet the requirement of routine analysis and quality control and evaluation.

[Determination of 8 components in healthy food for anti-hangover and hepatoprotection by high performance liquid chromatography].

OBJECTIVE: To develop a simple and sensitive high performance liquid chromatographic method for simultaneous determination of catechin hydrate, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, dihydromyricetin, glycyrrhizic acid and glycyrrhetinic acid in healthy food for anti-hangover and hepatoprotection, and compare with the capillary electrophoresis method established by our laboratory. METHODS: The samples were ultrasonically extracted by using methanol-water( 4∶ 1, V/V) for 30 minutes and then centrifuged at 10 000 r/min for 10 minutes. The supernatant was filtered and injected into the HPLC system and then separated on a C_(18) column( 5 µm × 250 mm × 4. 6 mm) at 30℃ with gradient elution at a flow rate of 0. 8mL/min. Catechins and dihydromyricetin were detected at the wavelength of 210 nm, glycyrrhizic acid and glycyrrhetinic acid were detected at 250 nm. RESULTS: Under the optimal analytical conditions, the peak area of each analyte and its concentration had agood correlation within the linear range( r ≥ 0. 9996). The limits of detection and quantification of the method were 0. 07-1. 25 µg/g( S/N = 3) and 0. 22-4. 18 µg/g( S/N = 10), respectively. The intra-and inter-day relative standard deviations( RSDs)of the mixed standard solution were 0. 26%-1. 95% and 1. 17%-3. 89%, respectively. The spiked recoveries of the analytes were 86. 15%-98. 61%. CONCLUSION: The established method is sensitive and reliable, and could be used for quality control of the healthy food for anti-hangover and hepatoprotection.

Identification of Vibrio cholerae serotypes in high-risk marine products with non-gel sieving capillary electrophoresis.

Vibrio cholerae, a natural inhabitant of the marine environment, poses a threat to human health, and its new epidemic variants have been reported. A method of multiplex polymerase chain reaction-capillary electrophoresis-laser-induced fluorescence (PCR-CE-LIF) detection has been developed to detect and identify V. cholerae in marine products sensitively, rapidly, and reliably. Four sets of primers were selected to amplify genus-specific VCC gene, O139 serogroup-specific O139 gene, O1 serogroup-specific O1 gene, and ctxA gene associated with the CT toxin of enterotoxigenic V. cholerae. The PCR products were detected using CE-LIF with SYBR Gold serving as the DNA fluorescent dye. The parameters of PCR and the separation conditions of CE-LIF were optimized. Under the optimal conditions, V. cholerae was detected and four serotypes were identified simultaneously within 8 min. The alignment analysis showed that the PCR products had good agreement with the published sequences from GenBank, indicating that the primers selected in this study had high specificity and the PCR results were reliable. The proposed method could detect 5 to 20 cfu/ml V. cholerae. The intraday precisions of migration time and peak area of DNA marker and PCR products were in the ranges of 1.60-2.56% and 1.60-6.29%, respectively. The specificity results showed that only five standard bacteria used in this study showed the specific peaks when the target bacteria were mixed with seven other common intestinal pathogenic bacteria at the same concentration. The assay was applied to 71 high-risk marine products, and different serotypes of V. cholerae could be identified sensitively and reliably.