Triblock PolyA-Mediated Protein Biosensor Based on a Size-Matching Proximity Hybridization Analysis.

The antibodies in the natural biological world utilize bivalency/multivalency to achieve a higher affinity for antigen capture. However, mimicking this mechanism on the electrochemical sensing interface and enhancing biological affinity through precise spatial arrangement of bivalent aptamer probes still pose a challenge. In this study, we have developed a novel self-assembly layer (SAM) incorporating triblock polyA DNA to enable accurate organization of the aptamer probes on the interface, constructing a "lock-and-key-like" proximity hybridization assay (PHA) biosensor. The polyA fragment acts as an anchoring block with a strong affinity for the gold surface. Importantly, it connects the two DNA probes, facilitating one-to-one spatial proximity and enabling a controllable surface arrangement. By precisely adjusting the length of the polyA fragment, we can tailor the distance between the probes to match the molecular dimensions of the target protein. This design effectively enhances the affinity of the aptamers. Notably, our biosensor demonstrates exceptional specificity and sensitivity in detecting PDGF-BB, as confirmed through successful validation using human serum samples. Overall, our biosensor presents a novel and versatile interface for proximity assays, offering a significantly improved surface arrangement and detection performance.

Construction of an Enzyme Cascade Based on the Accurate Adjacent Arrangement of Coupled Enzymes Using a Triblock PolyA DNA Probe.

Intracellular enzyme cascades are essential for various biological processes, and mimicking their functions in artificial systems has attracted significant research attention. However, achieving convenient and efficient spatial organization of enzymes on interfaces remains a critical challenge. In this work, we designed a simple single-DNA scaffold using triblock polyA single-stranded DNA for the arrangement of coupled enzymes. The scaffold was assembled onto a gold electrode through the affinity of polyA-Au, and two enzymes (glucose oxidase and horseradish peroxidase) were captured through hybridization. The molecular distance between the enzymes was regulated by changing the length of the polyA fragment. As a proof of concept, a glucose biosensor was constructed based on the enzyme cascade amplification. The biosensor exhibited excellent detection capability for glucose in human serum samples with a limit of detection of 1.6 µM. Additionally, a trienzyme cascade reaction was successfully activated, demonstrating the potential scalability of our approach for multienzyme reactions. This study provides a promising platform for the development of easy-to-operate, highly efficient, and versatile enzyme cascade systems using DNA scaffolds.

Multi-omics data integration using ratio-based quantitative profiling with Quartet reference materials.

Characterization and integration of the genome, epigenome, transcriptome, proteome and metabolome of different datasets is difficult owing to a lack of ground truth. Here we develop and characterize suites of publicly available multi-omics reference materials of matched DNA, RNA, protein and metabolites derived from immortalized cell lines from a family quartet of parents and monozygotic twin daughters. These references provide built-in truth defined by relationships among the family members and the information flow from DNA to RNA to protein. We demonstrate how using a ratio-based profiling approach that scales the absolute feature values of a study sample relative to those of a concurrently measured common reference sample produces reproducible and comparable data suitable for integration across batches, labs, platforms and omics types. Our study identifies reference-free 'absolute' feature quantification as the root cause of irreproducibility in multi-omics measurement and data integration and establishes the advantages of ratio-based multi-omics profiling with common reference materials.

Development of a Single-Molecule Nanobalance Ratiometric Electrochemical DNA Biosensor Using a Triblock Poly-Adenine Probe.

The development of electrochemical DNA biosensors has been limited by their reliability and reproducibility due to many interfering factors such as electrode properties, DNA surface densities, and complex biological samples. In this work, we developed a nanobalance polyA hairpin probe (polyA-HP), which was effectively assembled onto the gold electrode surface through the affinity between the central polyA fragment and the Au surface. One flanking probe of the polyA-HP captured the target sequence together with a MB-labeled signal probe, and the other flanking probe captured a reference probe simultaneously. The MB signal related to the amount of target was normalized by the reference Fc signal; thus, the signal-to-noise (S/N) was as high as 2000, and the reproducibility was remarkably improved to 2.77%, even facing deliberately changed experiment conditions. By designing a hairpin structure at the terminal of the polyA-HP, the selectivity and specificity were dramatically improved for the analysis of mismatched sequences. The analysis performance of biological samples was dramatically improved after normalization, which is critical for its practicability. Our novel biosensor is a universal single-molecule platform for ratiometric biosensors with excellent performance in real samples, indicating great potential for next-generation high-precision electrochemical sensors.

Triblock probe-polyA-probe electrochemical interfacial engineering for the sensitive analysis of RNAi plants.

RNA interference (RNAi) is currently under fast development, which brings improved crop quality and new activity against pests in agriculture, by producing RNAs to specifically inhibit gene expression. This technology, in turn, creates a pressing need for sensitive and specific analytical methods of exogenous RNA molecules in genetically modified (GM) crops for safety assessment and regulation of RNAi plants and their products. In this work, we developed a novel RNA electrochemical biosensor for the analysis of GM maize samples based on a polyA-DNA capturing probe containing three DNA segments: the central polyA segment combined onto a gold electrode surface with adjustable configuration and density, and two flanking DNA probes simultaneously captured the RNA targets through hybridization. Both the assembling and hybridization capability of our probe were demonstrated, and we systematically optimized the analytical conditions. Finally, the ultrasensitive detection of 10 fM RNA was realized without any amplification processes, and the specificity was verified by analyzing non-target maize samples. Our electrochemical biosensor provided a reliable and convenient measurement strategy for RNAi safety and quality assessment, and more importantly, our PAP (probe-polyA-probe) capturing probe exhibited an innovative design for the detection of large RNA molecules with complex secondary structures.

Determining the rotational alignment of the tibial component referring to the tibial tubercle during total knee arthroplasty: the tibial tubercle-trochlear groove can be an aid.

BACKGROUND: There is no consensus on anatomic landmarks or reference axes with which to accurately align rotational position of tibial component. Using the tibial tubercle, commonly referring to the Akagi line and the Insall line, for anatomic reference was widely accepted. However, it is unknown about the predictors that may affect the reliability of using the tibial tubercle for aligning tibial component rotation. The aims of our study were (1) to investigate the reproducibility and accuracy of using the tibial tubercle for aligning tibial component rotation and (2) to determine predictors resulting in discrepancies of the tibial component rotation when referring to the tibial tubercle. METHOD: A total of 160 patients with osteoarthritis were recruited before total knee arthroplasty. The angle α formed by the tibial anteroposterior (AP) axis and the Akagi line and the angle ß formed by the tibial AP axis and the Insall line were measured to quantify the discrepancies of the Akagi line and the Insall line. Independent variables, including the tibial tubercle-to-trochlear groove distance (TT-TG), tibial tubercle to posterior cruciate ligament (TT-PCL), and knee rotation angle (KRA), hip-knee-ankle angle (HKA), medial proximal tibial angle (MPTA), and tibial bowing (TB), were measured. Pearson's product moment correlation coefficients and multivariable linear regression analysis were calculated to assess relationships between independent variables and the two defined angles. RESULTS: All defined measurement were available for 140 patients. The Akagi line rotated internally with 1.03° ± 4.25° in regard to the tibial AP axis. The Insall line rotated externally in regard to the tibial AP axis with 7.93° ± 5.36°. Three variables, including TT-TG, TT-PCL, and KRA, tended to be positively correlated with the angle α and the angle ß. In terms of a cutoff of TT-TG = 9 mm, 100% cases and 97% cases for using the Akagi line and Insall line, respectively, were located in the defined safe zone (- 5° to 10°). CONCLUSION: The tibial tubercle (the Akagi line and Insall line) is found to be a useful and promising anatomic landmark for aligning the tibial component rotation. The TT-TG, with a cutoff value of 9 mm, is helpful to choose the Akagi line or Insall line, alternatively.

Effects of 7S and 11S on the intestine of weaned piglets after injection and oral administration of soybean antigen protein.

Soybeans are used increasingly in food products because of their health benefits. In this study, we investigated the effect of soybean antigen protein on weaned piglet intestine. Seventy piglets were randomly divided into seven groups with 10 piglets each. At 7 and 14 days of age, groups A-C were injected with saline, and D-G were intramuscularly injected with or orally administered 7S or 11S. Groups B-G were artificially sensitized by dietary 7S or 11S. At 27 days, the small intestinal tissues were collected to determine levels of histamine, sIgA protein, and IgA mRNA. Histamine in B-G was significantly decreased in the duodenum and ileum. Moreover, sIgA expression was higher in all groups than in A, with B/C>D-G and F/G>D/E; the trend in IgA expression was similar. Collectively, these results indicated that soybean antigen protein-immunizing agents decrease sIgA and IgA levels. Additionally, the effect of injection immunization occurred prior to that of oral immunization.

Soybean Glycinin- and ß-Conglycinin-Induced Intestinal Damage in Piglets via the p38/JNK/NF-κB Signaling Pathway.

ß-Conglycinin (7S) and glycinin (11S) are known to induce a variety of hypersensitivity reactions involving the skin, intestinal tract, and respiratory tract. The present study aimed to identify the mechanism underlying the development of allergy to soybean antigen proteins, using piglets as an animal model. Weaned "Duroc × Landrace × Yorkshire" piglets were fed a diet supplemented with 7S or 11S to investigate the signaling pathway involved in intestinal damage in piglets. Results showed that serum nitric oxide (NO), tumor necrosis factor-α (TNF-α), and caspase-3 levels were significantly higher in 7S- and 11S-fed piglets compared to those in suckling or weaned ones. mRNA, protein, and phosphorylation levels of nuclear factor-kappa B (NF-κB), p38, and Jun N-terminal kinase (JNK) were higher in 7S- and 11S-fed piglets than in suckling and weaned ones. Overall, our results indicate that 7S and 11S damaged the intestinal function in piglets through their impact on NF-κB, JNK, and p38 expression.

Intermediate-Temperature Creep Deformation and Microstructural Evolution of an Equiatomic FCC-Structured CoCrFeNiMn High-Entropy Alloy.

The tensile creep behavior of an equiatomic CoCrFeNiMn high-entropy alloy was systematically investigated over an intermediate temperature range (500-600 °C) and applied stress (140-400 MPa). The alloy exhibited a stress-dependent transition from a low-stress region (LSR-region I) to a high-stress region (HSR-region II). The LSR was characterized by a stress exponent of 5 to 6 and an average activation energy of 268 kJ mol-1, whereas the HSR showed much higher corresponding values of 8.9-14 and 380 kJ mol-1. Microstructural examinations on the deformed samples revealed remarkable dynamic recrystallization at higher stress levels. Dislocation jogging and tangling configurations were frequently observed in LSR and HSR at 550 and 600 °C, respectively. Moreover, dynamic precipitates identified as M23C6 or a Cr-rich σ phase were formed along grain boundaries in HSR. The diffusion-compensated strain rate versus modulus-compensated stress data analysis implied that the creep deformation in both stress regions was dominated by stress-assisted dislocation climb controlled by lattice diffusion. Nevertheless, the abnormally high stress exponents in HSR were ascribed to the coordinative contributions of dynamic recrystallization and dynamic precipitation. Simultaneously, the barriers imposed by these precipitates and severe initial deformation were referred to so as to increase the activation energy for creep deformation.

Electrochemical detection of PCR amplicons of Escherichia coli genome based on DNA nanostructural probes and polyHRP enzyme.

Fast, portable and sensitive analysis of E. coli is becoming an important challenge in many critical fields (e.g., food safety, environmental monitoring and clinical diagnosis). Thus, electrochemical biosensing of PCR amplicons from the bacterial genome has attracted reasonable research attention. In this work, we utilized a 3D DNA tetrahedral probe to establish a "sandwich-type" electrochemical DNA biosensor for sensitive and specific analysis of a 250 bp unpurified PCR amplicon from the uidA gene of the E. coli genome. Asymmetric PCR was used to produce single-stranded PCR products. Streptavidin-polyHRP80 was employed to improve the signal gain during electrochemical detection. We optimized important experimental conditions for DNA sensing, including the streptavidin-polyHRP, the signal probe and the ion strength. Finally, we achieved a remarkable sensitivity of 10 fM synthetic DNA target, and successfully performed the analysis of PCR amplicons from as low as 0.2 pg µL(-1) of E. coli genome. Compared with traditional single stranded DNA (ssDNA) probe based detection, our present work demonstrated 3 orders of magnitude improvement in sensitivity. In addition, our electrochemical DNA biosensor was 4 orders of magnitude more sensitive than normal electrophoretic analysis of PCR products. Our work made important progress in DNA nanostructured probe-based biosensors toward application in real applications.