A 3D-Printed Integrated Handheld Biosensor for the Detection of Vibrio parahaemolyticus.

Vibrio parahaemolyticus (V. parahaemolyticus) is one of the important seafood-borne pathogens that cause a serious gastrointestinal disorder in humans. Recently, biosensors have attracted serious attention for precisely detecting and tracking risk factors in foods. However, a major consideration when fabricating biosensors is to match the low cost of portable devices to broaden its application. In this study, a 3D-printed integrated handheld biosensor (IHB) that combines RPA-CRISPR/Cas12a, a lateral flow strip (LFS), and a handheld device was developed for the ultrasensitive detection of V. parahaemolyticus. Using the preamplification of RPA on tlh gene of V. parahaemolyticus, a specific duplex DNA product was obtained to activate the trans-cleavage activity of CRISPR/Cas12a, which was then utilized to cleave the ssDNA probe. The ssDNA probe was then detected by the LFS, which was negatively correlated with the content of amplified RPA products of the tlh gene. The IHB showed high selectivity and excellent sensitivity for V. parahaemolyticus detection, and the limit of detection was 4.9 CFU/mL. The IHB also demonstrated great promise for the screening of V. parahaemolyticus in samples and had the potential to be applied to the rapid screening of other pathogen risks for seafood and marine environmental safety.

Ecotoxicological QSAR study of fused/non-fused polycyclic aromatic hydrocarbons (FNFPAHs): Assessment and priority ranking of the acute toxicity to Pimephales promelas by QSAR and consensus modeling methods.

Fused/non-fused polycyclic aromatic hydrocarbons (FNFPAHs) have a variety of toxic effects on ecosystems and human body, but the acquisition of their toxicity data is greatly limited by the limited resources available. Here, we followed the EU REACH regulation and used Pimephales promelas as a model organism to investigate the quantitative structure-activity relationship (QSAR) between the FNFPAHs and their toxicity for the aquatic environment for the first time. We developed a single QSAR model (SM1) containing five simple and interpretable 2D molecular descriptors, which met the validation of OECD QSAR-related principles, and analyzed their mechanistic relationships with toxicity in detail. The model had good degree of fitting and robustness, and had better external prediction performance (MAEtest = 0.4219) than ECOSAR model (MAEtest = 0.5614). To further enhance its prediction accuracy, the three qualified single models (SMs) were used for constructing consensus models (CMs), the best one CM2 (MAEtest = 0.3954) had a significantly higher prediction accuracy for test compounds than SM1, and also outperformed the T.E.S.T. consensus model (MAEtest = 0.4233). Subsequently, the toxicity of 252 true external FNFPAHs from Pesticide Properties Database (PPDB) was predicted by SM1, the prediction results showed that 94.84 % compounds were reliably predicted within the model's application domain (AD). We also applied the best CM2 to predict the untested 252 FNFPAHs. Furthermore, we provided a mechanistic analysis and explanation for pesticides ranked as top 10 most toxic FNFPAHs. In summary, all developed QSAR and consensus models can be used as efficient tools for predicting the acute toxicity of unknown FNFPAHs to Pimephales promelas, thus being important for the risk assessment and regulation of FNFPAHs contamination in aquatic environment.

Protection of DNA by metal ions at 95 °C: from lower critical solution temperature (LCST) behavior to coordination-driven self-assembly.

While polyvalent metal ions and heating can both degrade nucleic acids, we herein report that a combination of them leads to stabilization. After incubating 4 mM various metal ions and DNA oligonucleotides at 95 °C for 3 h at pH 6 or 8, metal ions were divided into four groups based on gel electrophoresis results. Mg2+ can stabilize DNA at pH 6 without forming stable nanoparticles at room temperature. Co2+, Cu2+, Cd2+, Mn2+ and Zn2+ all protected the DNA and formed nanoparticles, whereas the nanoparticles formed with Fe2+ and Ni2+ were so stable that they remained even in the presence of EDTA. At pH 8, Ce3+ and Pb2+ showed degraded DNA bands. For Mg2+, better protection was achieved with higher metal and DNA concentrations. By monitoring temperature-programmed fluorescence change, a sudden drop in fluorescence intensity attributable to the lower critical solution temperature (LCST) transition of DNA was found to be around 80 °C for Mg2+, while this transition temperature decreased with increasing Mn2+ concentration. The unexpected thermal stability of DNA enabled by metal ions is useful for extending the application of DNA at high temperatures, forming coordination-driven nanomaterials, and it might offer insights into the origin of life on the early Earth.

Chemopreventive Role of Apigenin against the Synergistic Carcinogenesis of Human Papillomavirus and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Tobacco smoke and human papillomavirus (HPV) are both crucial causes of cancer, and their cooperative carcinogenesis has drawn more attention in recent years. Apigenin (AP), a typical flavonoid abundantly found in flowers of plants, vegetables, and fruits, has been demonstrated to exert an anti-carcinogenic effect on various types of cancer. In this study, we investigated the capability of AP against malignant transformation and DNA damage of immortalized human esophageal epithelial (SHEE) cells induced by the synergism of HPV18 and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The results indicated that the enhancement of migration, invasion, and proliferation ability of SHEE cells induced by HPV and NNK could be effectively inhibited by AP. Moreover, the levels of pyridyloxybutylated (POB)-DNA adducts induced by NNK via P450-catalyzed metabolic activation could also be significantly suppressed by AP. Further analyses on the molecular mechanism revealed that AP inhibited the synergistic carcinogenesis of NNK and HPV on SHEE cells by reducing the expression of mutp53, CDK4, Cyclin D1, and p-Rb (Ser 780), increasing caspase-3 activity, thereby arresting the cell cycle at G1 phase and promoting apoptosis of SHEE cells. We hypothesize that the decrease in NNK-induced POB-DNA adduct levels is related to the deactivation of P450 by AP, which needs to be confirmed in future studies. This study highlights that AP may be employed as a promising chemopreventive agent against cancers in smokers with an HPV infection.

A 'turn-on' ultra-sensitive multiplex real-time fluorescent quantitative biosensor mediated by a universal primer and probe for the detection of genetically modified organisms.

Among the existing multiplex genetically modified organism (GMO) detection methods, significant problems are highlighted, including amplification asymmetry of different targets, and the low detection throughput, which limits their capacity to meet the requirements of high-throughput analysis. To mitigate these challenges, a 'turn-on' ultra-sensitive multiplex real-time fluorescent quantitative biosensor is developed. In this system, the multiplex ligation-dependent amplification (MLPA), universal primer and universal probe are innovatively combined, which can enhanced the amplification specificity, overcome asymmetric amplification and guarantee the homogeneity of amplification efficiency simultaneously. Furthermore, both single and multiplex detection results can be output by the fluorescent group labeled on universal TaqMan probes for different targets in real-time. After optimization, the quantitative detection limit was 5 pg. In conclusion, this strategy could serve as an important tool for GMO detection in processed and commercially available products, even in the fields that require reliable and sensitive detection of DNA targets.

Multiplex pyrosequencing quantitative detection combined with universal primer-multiplex-PCR for genetically modified organisms.

A multiplex pyrosequencing quantitative detection technique combined with universal primer-multiplex-PCR (UP-M-PCR) was established. In this study, a pyrosequencing results analysis software was first self-compiled, which realized the DNA sequences degeneration, and converted the pyrosequencing results and base composition of the target sequences into mathematic relations. Five calculation models were put forward based on the actual situation, which adjusted the values smaller than zero or the detection limit. By applying this method, samples containing five genetically modified (GM) lines mixed in random ratio were quantified, it showed that the quantification was very close to the actual value, and the detection sensitivity was as low as 1.47% of a single component, which satisfied most labeling policies. This novel method is realized without fluorescent group labeling, hence the number of targets is not limited by factors inherent in method or equipment, and is proven to be a reliable tool for the quantitative detection.

Freezing promoted hybridization of very short DNA oligonucleotides.

Shorter DNA probes provide better specificity for hybridization, but they may not form stable duplexes at room temperature. In this study, we used thiazole orange to follow DNA hybridization upon freezing and achieved stable 5-mer duplex DNA. Using multiple short probes in tandem, long DNA could also be studied. This study provides insights into DNA hybridization in the frozen state and expands the application of freezing for nucleic acid chemistry.

Rapid and low-cost strategy for detecting genome-editing induced deletion: A single-copy case.

Genome editing techniques have been implemented in human daily lives, which has created a high demand for the development of new gene-edited product analysis methods. Conventional assays are time-consuming, labor-intensive, and costly. This paper proposes a rapid and low-cost strategy for detecting genome-editing induced deletion which works by integrating rapid-multiplex ligation-dependent probe amplification (MLPA) with a dual-lateral flow nucleic acid biosensor (LFNAB) cascade in a single-copy case. A rapid-MLPA was first introduced to the LFNAB system as a replacement for the conventional PCR for enhanced specificity and accuracy. A dual-LFNAB was applied for the detection of genome-editing induced deletion without any additional instrumentation or complex operation. After optimization, we achieved the specific detection of wildtype alleles and deletion alleles in spiked samples with a detection limit of 0.4 fM, which is comparable to that of electrophoresis-based detection assays and fluorescent biosensors. To confirm the validity and feasibility of our strategy, we assayed two pork samples from two WUZHISHAN pigs successfully. By comparing the detection results from next-generation sequencing analysis, we found that the proposed cascade demonstrates at least 20-fold shorter assay time and at least 100-fold less assay cost. To this effect, the proposed method is a rapid and low-cost solution to sample-to-answer detection of genome-editing induced deletion and shows remarkable potential in regards to international trade, transparency, and freedom of choice.

Ultrasensitive Single Fluorescence-Labeled Probe-Mediated Single Universal Primer-Multiplex-Droplet Digital Polymerase Chain Reaction for High-Throughput Genetically Modified Organism Screening.

As genetically modified (GM) technology develops and genetically modified organisms (GMOs) become more available, GMOs face increasing regulations and pressure to adhere to strict labeling guidelines. A singleplex detection method cannot perform the high-throughput analysis necessary for optimal GMO detection. Combining the advantages of multiplex detection and droplet digital polymerase chain reaction (ddPCR), a single universal primer-multiplex-ddPCR (SUP-M-ddPCR) strategy was proposed for accurate broad-spectrum screening and quantification. The SUP increases efficiency of the primers in PCR and plays an important role in establishing a high-throughput, multiplex detection method. Emerging ddPCR technology has been used for accurate quantification of nucleic acid molecules without a standard curve. Using maize as a reference point, four heterologous sequences ( 35S, NOS, NPTII, and PAT) were selected to evaluate the feasibility and applicability of this strategy. Surprisingly, these four genes cover more than 93% of the transgenic maize lines and serve as preliminary screening sequences. All screening probes were labeled with FAM fluorescence, which allows the signals from the samples with GMO content and those without to be easily differentiated. This fiveplex screening method is a new development in GMO screening. Utilizing an optimal amplification assay, the specificity, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The LOD and LOQ of this GMO screening method were 0.1% and 0.01%, respectively, with a relative standard deviation (RSD) < 25%. This method could serve as an important tool for the detection of GM maize from different processed, commercially available products. Further, this screening method could be applied to other fields that require reliable and sensitive detection of DNA targets.

Nucleic Acid Biosensor Synthesis of an All-in-One Universal Blocking Linker Recombinase Polymerase Amplification with a Peptide Nucleic Acid-Based Lateral Flow Device for Ultrasensitive Detection of Food Pathogens.

In this study, a whole-course nucleic-acid-constructed biosensor that combines the all-in-one concepts of the universal blocking linker recombinase polymerase amplification (UBLRPA) and a peptide nucleic acid (PNA)-based lateral flow device (PLFD) has been developed for the ultrasensitive detection of food pathogens. Using the preamplification UBLRPA principle, a universal linker and C3 space blocker were utilized to produce the universal linker single-duplex DNA products. The developed amplification system was employed to convert duplex products to a single strand. In the signal recognition strategy, a special PNA probe was successfully employed in the portable PLFD. The UBLRPA products were identified visually using the PLFD through dual hybridization (a PNA probe on the gold nanoparticle (Au-NP) was combined with a universal linker on the end of the products; a PNA capture probe was used on the test line and a universal linker on the other end of the products). The accumulation of Au-NPs produced a characteristic red band, enabling the visual detection of a food pathogen without further testing. To demonstrate the value of the all-in-one biosensor, Salmonella enterica subsp. enterica serovar typhimurium was used as a model organism. The biosensor showed high selectivity and extraordinary repeatability using S. typhimurium, and the limit of detection was 4 CFU mL-1. Furthermore, when milk samples artificially contaminated with S. typhimurium were analyzed, the analysis was completed within 30 min without complicated instrumentation. The results exhibited good precision and recovery. This portable all-in-one biosensor demonstrates great promise for the screening of pathogens in food and environmental samples.

Colorimetric biosensor based on a DNAzyme primer and its application in logic gate operations for DNA screening.

A colorimetric biosensor for DNA screening was designed based on the conformational changes of the horseradish peroxidase (HRP)-mimicking DNAzyme. The scheme of DNA biosensing was designed based on the base pairing of DNAzyme sequence to inhibit the formation of HRP-mimicking hemin/G-quadruplex structures in the process of amplification. DNA could be amplified via the universal primer multiplex polymerase chain reaction (UP-M-PCR) and innovatively detected as color disappear in the reaction visible to the naked eye. The input of key factors and the output of optical characteristics in the reaction inspired the development of an OR logic gate operation for DNA detection. This biosensor overcomes self-inhibition and amplification disparity with the help of UP-M-PCR, thereby exhibiting high specificity and high-throughput without the requirement of gel analysis work. This biosensing system also presented 1% sensitivity and approximately 180 copy numbers in triplicate. The biosensor was used to screen elements from genetically modified organisms (GMOs) and covered more than 90% of all globally authorized events in the world. The designed colorimetric biosensor is a rapid, portable and versatile tool for nucleic acids detection and diagnosis in the field.

Ultrasensitive Detection of Viable Enterobacter sakazakii by a Continual Cascade Nanozyme Biosensor.

Recent outbreaks of life-threatening neonatal infections linked to Enterobacter sakazakii (ES) heightened the need to develop rapid and ultrasensitive detection strategies, especially those capable of determining the viable cells. This study introduced a continual cascade nanozyme biosensor for the detection of viable ES based on propidium monoazide (PMA), loop-mediated isothermal amplification (LAMP), and Nanozyme strip. The ompA gene of ES was determined using FITC-modified and BIO-modified primers in the LAMP process. LAMP combined with PMA treatment was applied for distinguishing the viable from the dead state of ES. Then, using Fe3O4 magnetic nanoparticles as a nanozyme probe, a magnetic nanoparticle (MNP)-based immunochromatographic strip (Nanozyme strip) was further employed for amplifying signal to allow visual detection and quantification by a strip reader. The LAMP products were sandwiched between the anti-FITC and the anti-BIO, and the accumulation of the Fe3O4 magnetic nanoparticles enabled the visual detection of ES. The detection limit of the nanozyme biosensor was improved by 10 CFU/mL compared with previously reported techniques, and the whole manipulation process was much faster (within 1 h) and simpler (without specialist facilities). Hence, the developed continual cascade nanozyme biosensor has provided a rapid, ultrasensitive, and simple tool for on-site detection of viable ES.

Novel multiplex qualitative detection using universal primer-multiplex-PCR combined with pyrosequencing.

This study described a novel multiplex qualitative detection method using pyrosequencing. Based on the principle of the universal primer-multiplex-PCR, only one sequencing primer was employed to realize the detection of the multiple targets. Samples containing three genetically modified (GM) crops in different proportions were used to validate the method. The dNTP dispensing order was designed based on the product sequences. Only 12 rounds (ATCTGATCGACT) of dNTPs addition and, often, as few as three rounds (CAT) under ideal conditions, were required to detect the GM events qualitatively, and sensitivity was as low as 1% of a mixture. However, when considering a mixture, calculating signal values allowed the proportion of each GM to be estimated. Based on these results, we concluded that our novel method not only realized detection but also allowed semi-quantitative detection of individual events.

Identification of a chicken (Gallus gallus) endogenous reference gene (Actb) and its application in meat adulteration.

The genes commonly used to determine meat species are mainly mitochondrial, but the copy numbers of such genes are high, meaning they cannot be accurately quantified. In this paper, for the first time, the chromosomal gene Actb was selected as an endogenous reference gene for chicken species. It was assayed in four different chicken varieties and 16 other species using both qualitative and quantitative PCR. No amplification of the Actb gene was found in species other than chicken and no allelic variations were detected in chicken. Southern blot and digital-PCR confirmed the Actb gene was present as a single copy in the chicken genome. The quantitative detection limit was 10pg of DNA, which is equivalent to eight copies. All experiments indicated that the Actb gene is a useful endogenous reference gene for chicken, and provides a convenient and accurate approach for detection of chicken in feed and food.

Specific and relative detection of urinary microRNA signatures in bladder cancer for point-of-care diagnostics.

We present a dual-isothermal cascade strategy assisted by a lateral flow peptide nucleic acid biosensor for point-of-care detection of urinary microRNAs without a temperature protocol and complex instruments. The proposed assay is expected to be of great promise for bladder cancer diagnosis and point-of-care diagnostics.

A smart sealed nucleic acid biosensor based on endogenous reference gene detection to screen and identify mammals on site.

The identification of meat adulteration is a hotspot for food research worldwide. In this paper, a smart and sealed biosensor that combines loop-mediated isothermal amplification (LAMP) with a lateral flow device (LFD) was developed, resulting in the universal mammalian assessment on site. First, the highly specific chromosomal Glucagon gene (Gcg) was chosen as the endogenous reference gene, and the LAMP approach provided double-labeled duplex DNA products using FITC- and BIO- modified primers. Then, an LFD strategy was used for specific signal recognition through an immunoassay. Meanwhile, LFD-LAMP was compared to LAMP and real-time LAMP, the results showed consistent high specificity and sensitivity but in a more convenient and easy-to-use system. In addition, the detection limit was as low as 10 pg, which was equivalent to 3~5 copies in mammals. All of the reactions were performed in a sealed system regardless of the amplification process or products recognized. Therefore, the smart design demonstrated significantly high specificity and the ability to detect trace amounts of DNA in complex and processed foods with mammalian meat. As a universal and specific platform for the detection of mammalian DNA, this smart biosensor is an excellent prospect for species identification and meat adulteration.

On-site detection of stacked genetically modified soybean based on event-specific TM-LAMP and a DNAzyme-lateral flow biosensor.

Stacked genetically modified organisms (GMO) are becoming popular for their enhanced production efficiency and improved functional properties, and on-site detection of stacked GMO is an urgent challenge to be solved. In this study, we developed a cascade system combining event-specific tag-labeled multiplex LAMP with a DNAzyme-lateral flow biosensor for reliable detection of stacked events (DP305423× GTS 40-3-2). Three primer sets, both event-specific and soybean species-specific, were newly designed for the tag-labeled multiplex LAMP system. A trident-like lateral flow biosensor displayed amplified products simultaneously without cross contamination, and DNAzyme enhancement improved the sensitivity effectively. After optimization, the limit of detection was approximately 0.1% (w/w) for stacked GM soybean, which is sensitive enough to detect genetically modified content up to a threshold value established by several countries for regulatory compliance. The entire detection process could be shortened to 120min without any large-scale instrumentation. This method may be useful for the in-field detection of DP305423× GTS 40-3-2 soybean on a single kernel basis and on-site screening tests of stacked GM soybean lines and individual parent GM soybean lines in highly processed foods.

One-step competitive lateral flow biosensor running on an independent quantification system for smart phones based in-situ detection of trace Hg(II) in tap water.

In this study, a one-step lateral flow biosensor (LFB) has been developed, optimized and validated for quantitative detection of Hg(II) in water. In the measurement principle, just one T-rich ssDNA probe (TSP) for the specific binding process was successfully employed in the competitive LFB based methods. The concept of an independent quantification system was realized using a cresol red dot as an external standard, which effectively eliminates false negative results. Under optimized conditions, the limit of detection for Hg(II) was 4nM; high selectivity towards Hg(II) and extraordinary device-to-device repeatability of the LFB were achieved. Furthermore, Hg(II) from tap water samples was analyzed, and the results were confirmed by ICP-MS. The interference from other components in the real samples could be neglected during the analysis. The approach provides a simple, sensitive, and practical tool for the detection of trace Hg(II) in tap water, showing great promise for in-situ applications.

High-sensitivity assay for Hg (II) and Ag (I) ion detection: A new class of droplet digital PCR logic gates for an intelligent DNA calculator.

The first example of droplet digital PCR logic gates ("YES", "OR" and "AND") for Hg (II) and Ag (I) ion detection has been constructed based on two amplification events triggered by a metal-ion-mediated base mispairing (T-Hg(II)-T and C-Ag(I)-C). In this work, Hg(II) and Ag(I) were used as the input, and the "true" hierarchical colors or "false" green were the output. Through accurate molecular recognition and high sensitivity amplification, positive droplets were generated by droplet digital PCR and viewed as the basis of hierarchical digital signals. Based on this principle, YES gate for Hg(II) (or Ag(I)) detection, OR gate for Hg(II) or Ag(I) detection and AND gate for Hg(II) and Ag(I) detection were developed, and their sensitively and selectivity were reported. The results indicate that the ddPCR logic system developed based on the different indicators for Hg(II) and Ag(I) ions provides a useful strategy for developing advanced detection methods, which are promising for multiplex metal ion analysis and intelligent DNA calculator design applications.

Real-time quantitative nicking endonuclease-mediated isothermal amplification with small molecular beacons.

Techniques of isothermal amplification have recently made great strides, and have generated significant interest in the field of point-of-care detection. Nicking endonuclease-mediated isothermal amplification (NEMA) is an example of simple isothermal technology. In this paper, a real-time quantitative nicking endonuclease-mediated isothermal amplification with small molecular beacons (SMB-NEMA) of improved specificity and sensitivity is described. First, we optimized the prohibition of de novo synthesis by choosing Nt·BstNBI endonuclease. Second, the whole genome was successfully amplified with Nt·BstNBI (6 U), betaine (1 M) and trehalose (60 mM) for the first time. Third, we achieved 10 pg sensitivity for the first time after adding a small molecular beacon that spontaneously undergoes a conformational change when hybridizing to target, and the practical test validated the assay's application. The small molecular beacon has a similar melting temperature to the reaction temperature, but is approximately 10 bp shorter than the length of a traditional molecular beacon. A new threshold regulation was also established for isothermal conditions. Finally, we established a thermodynamic model for designing small molecular beacons. This multistate model is more correct than the traditional algorithm. This theoretical and practical basis will help us to monitor SMB-NEMA in a quantitative way. In summary, our SMB-NEMA method allows the simple, specific and sensitive assessment of isothermal DNA quantification.