Fluorometric determination of microRNA by using an entropy-driven three-dimensional DNA walking machine based on a catalytic hairpin assembly reaction on polystyrene microspheres.

An entropy-driven 3-D DNA walking machine is presented which involves catalytic hairpin assembly (CHA) for detection of microRNA. A 3-D DNA walking machine was designed that uses streptavidin-coated polystyrene microspheres as track carriers to obtain reproducibility. The method was applied to microRNA 21 as a model analyte. Continuous walking on the DNA tracks is achieved via entropy increase. This results in a disassembly of ternary DNA substrates on polystyrene microspheres and leads to cycling of microRNA 21. The release of massive auxiliary strands from ternary DNA substrates induces the CHA. This is accompanied by in increase in fluorescence, best measured at excitation/emission wavelengths of 480/520 nm. On account of entropy-driven reaction, the assay is remarkably selective. It can differentiate microRNA 21 from homologous microRNAs in giving a signal that is less than 5% of the signal for microRNA 21 except for microRNA-200b. The assay works in the 50 pM to 20 nM concentration range and has a 41 pM detection limit. The method displays good reproducibility (between 1.1 and 4.2%) and recovery (from 99.8 to 104.0%). Graphical abstract An entropy-driven 3-D DNA walking machine is described. It is based on the use of polystyrene microspheres and of a catalytic hairpin assembly reaction for sensitive microRNA detection. Figure Notes: AS represents auxiliary strand; S represents substrate strand; LS represents link strand; F represents fuel nucleic acid; RepF represents nucleic acid labeled with FAM; RepQ represents nucleic acid labeled with BHQ1.

Electrochemical competitive immunodetection of messenger RNA modified with N6-methyladenosine by using DNA-modified mesoporous PtCo nanospheres.

N6-Methyladenosine (m6A) is the most abundant RNA modification in eukaryotic messenger RNA (mRNA). A highly sensitive electrochemical immunosensor is described for the determination of m6A-RNA. The method is based on the use of antibody (anti-m6A) and PtCo mesoporous nanospheres (MPNs). The analogously modified probe of type m6A-DNA-PtCo competes with m6A-RNA for antibodies on the gold electrode as an electrical signal probe. The electrical signal, best acquired at a working potential of -0.37 V (vs. Ag/AgCl) reflects the concentration of m6A. The PtCo MPNs catalyze the reduction of H2O2, and this amplifies the current and enhances sensitivity. The detection time of the assay is <1.5 h. Under optimal conditions, response is linear in the 0.005 to 100 nM m6A RNA concentration range, and the detection limit is 2.1 pM. The results obtained by this immunoassay with human cell lines are comparable to those obtained with a commercial kit. Graphical abstractSchematic representation of a method for electrochemical determination of m6A-modified mRNA. Anti-m6A Ab: antibody against m6A; BSA: bovine serum albumin; PtCo: PtCo mesoporous nanospheres; SH-m6A-DNA: DNA modified with both m6A and thiol groups; DPV: differential pulse voltammetry.

A triple-coated ligament graft to facilitate ligament-bone healing by inhibiting fibrogenesis and promoting osteogenesis.

Absence of ligament-bone healing due to poor bioactivity and hyperplasia of fibrous tissue caused by immune response severely impairs ligament grafts' functional duration in anterior cruciate ligament (ACL) reconstruction. While osteogenic modification is a popular technique for promoting ligament-bone integration, inadequate osseointegration remains a common experience, due to occupying fibrous hyperplasia and impaired osteogenesis potential. In the present study, a triple-nano-coating polyethylene terephthalate (PET) graft was developed by polydopamine self-assembly, chondroitin sulfate (CS) chemical-grafting and BMP-2 physical-immobilization to facilitate robust ligament-bone healing, The CS/polydopamine-modified PET (C-pPET) graft was demonstrated to inhibit fibrogenesis by regulating polarization of macrophages and promoting the secretion of anti-inflammatory factors. Moreover, the immunoregulatory function of CS cooperated with BMP-2 to facilitate osteogenic differentiation of stem cells, promoting the expression of ALP, Runx2, OCN and COL I. Bone regeneration was significantly enhanced at early-middle stage in the BMP-loaded pPET (B/pPET) group, while occurring at middle-late stage in the C-pPET group. Continuous new bone formation and optimal ligament-bone healing were observed in the B/C-pPET group via sequential and synergistic immune osteogenesis by CS and cytokine osteogenesis by BMP-2. Thus, the present study revealed a practical avenue for the promotion of ligament-bone healing through the development of a triple-nano-coating engineered ligament combining immunoregulatory anti-fibrogenesis and sequential-synergistic osteogenesis, which holds a great potential for improving the clinical efficacy of ligament graft in ACL reconstruction. STATEMENT OF SIGNIFICANCE: A triple-nano-coating polyethylene terephthalate (PET) graft was developed by polydopamine self-assembly, chondroitin sulfate (CS) chemical-grafting and BMP-2 physical-immobilization to facilitate robust ligament-bone healing. This study demonstrated that the multifunctional ligament grafts could reshape the local immune microenvironment by regulating macrophage phenotype and immune cytokine secretion to inhibit the fibrous hyperplasia and regulate stem cell towards osteogenic differentiation to promote bone regeneration. The present study demonstrates that efficient ligament-bone healing is achieved via the combination of immunoregulatory anti-fibrogenesis and dual osteogenesis of immunoregulation and cytokine induction.

[The progress in researches on biocompatibility for direct brain-machine interface].

An important application of the direct brain-machine interfaces are providing an outlet for severely paralyzed individuals to communicate with the world. According to different type of microelectrodes, brain-machine interfaces are divided into indirect-BMI and direct-BMI. Direct-BMI are intracortical recording devices designed to capture the action potentials of many individual neurons, especially those that code for movement or its intent. A key problem in research of BMI is how to enhance biocompatibility for direct-BMI. This review introduces some new microelectrodes of direct brain-machine interface which all have higher biocompatibility.

Effect of the Interposition of Calcium Phosphate Materials on Tendon-Bone Healing During Repair of Chronic Rotator Cuff Tear.

BACKGROUND: The current nature of tendon-bone healing after rotator cuff (RC) repair is still the formation of granulation tissue at the tendon-bone interface rather than the formation of fibrocartilage, which is the crucial structure in native tendon insertion and can be observed after knee ligament reconstruction. The interposition of calcium phosphate materials has been found to be able to enhance tendon-bone healing in knee ligament reconstruction. However, whether the interposition of these kinds of materials can enhance tendon-bone healing or even change the current nature of tendon-bone healing after RC repair still needs to be explored. HYPOTHESIS: The interposition of calcium phosphate materials during RC repair would enhance tendon-bone healing or change its current nature of granulation tissue formation into a more favorable process. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 144 male Sprague-Dawley rats underwent unilateral detachment of the supraspinatus tendon, followed by delayed repair after 3 weeks. The animals were allocated into 1 of 3 groups: (1) repair alone, (2) repair with Ca5(PO4)2SiO4 (CPS) bioceramic interposition, or (3) repair with hydroxyapatite (HA) bioceramic interposition at the tendon-bone interface. Animals were sacrificed at 2, 4, or 8 weeks postoperatively, and microcomputed tomography (micro-CT) was used to quantify the new bone formation at the repair site. New fibrocartilage formation and collagen organization at the tendon-bone interface was evaluated by histomorphometric analysis. Biomechanical testing of the supraspinatus tendon-bone complex was performed. Statistical analysis was performed using 1-way analysis of variance. Significance was set at P < .05. RESULTS: The micro-CT analysis demonstrated remarkable osteogenic activity and osteoconductivity to promote new bone formation and ingrowth of CPS and HA bioceramic, with CPS bioceramic showing better results than HA. Histological observations indicated that CPS bioceramic had excellent biocompatibility and biodegradability. At early time points after the RC repair, CPS bioceramic significantly increased the area of fibrocartilage at the tendon-bone interface compared with the control and HA groups. Moreover, CPS and HA bioceramics had significantly improved collagen organization. Biomechanical tests indicated that the CPS and HA groups have greater ultimate load to failure and stiffness than the control group at 4 and 8 weeks, and the CPS specimens exhibited the maximum ultimate load to failure, stiffness, and stress of the healing enthesis. CONCLUSION: Both CPS and HA bioceramics aid in cell attachment and proliferation and accelerate new bone formation, and CPS bioceramic has a more prominent effect on tendon-to-bone healing. CLINICAL RELEVANCE: Local application of CPS and HA bioceramic at the tendon-bone interface shows promise in improving healing after rotator cuff tear repair.

Medial patellofemoral ligament reconstruction using semitendinosus tendons: polyester suture augmentation versus nonaugmentation.

BACKGROUND: The comparative clinical outcome of medial patellofemoral ligament reconstruction (MPFLR) using semitendinosus tendons with and without polyester suture augmentation for recurrent patellar instability is unknown. HYPOTHESIS: Medial patellofemoral ligament reconstruction with polyester suture augmentation will yield better results than MPFLR without augmentation for recurrent patellar instability in adults. STUDY DESIGN: Randomized controlled trial; Level of evidence, 2. METHODS: One hundred patients with recurrent patellar instability receiving MPFLR using semitendinosus tendons were randomly divided into 2 groups either with or without polyester suture augmentation. Tibial tubercle transfer was performed in most cases. Follow-ups were performed at 12, 24, and 60 months postoperatively, and computed tomography (CT) was performed immediately after the operation and at follow-up. The passive patellar glide test was performed before surgery, immediately after MPFLR during the operation, and at each follow-up point. The degree of knee function was evaluated preoperatively and at 2 and 5 years postoperatively using the International Knee Documentation Committee, Lysholm, and Kujala rating scales. Redislocation or multiple episodes of patellar instability were considered failures. RESULTS: Forty-two patients in the augmentation group and 43 patients in the nonaugmentation group were followed for 5 years and received complete serial CT examinations and functional evaluations. The correction of the static patellar position deteriorated over time in the nonaugmentation group but not in the augmentation group. The results of the passive patellar glide test indicated stable patellae in all patients immediately after MPFLR and more stable patellae in the augmentation group at each follow-up point. Functional evaluations at 2 and 5 years revealed statistically significant superior results in the augmentation group. Finally, no patient in the augmentation group and 2 (4.7%) in the nonaugmentation group experienced episodes of redislocation, and 1 patient (2.4%) in the augmentation group and 8 (18.6%) in the nonaugmentation group experienced multiple episodes of patellar instability, resulting in failure rates of 2.4% and 23.3% in the augmentation group and nonaugmentation group, respectively (P = .004). CONCLUSION: Medial patellofemoral ligament reconstruction with polyester suture augmentation results in better static patellar position, dynamic stability, and functional outcome than without augmentation in the treatment of recurrent patellar dislocation in adults.

Determination of serum alcohol using a disposable biosensor.

This paper presents a disposable biosensor to detect serum alcohol concentration. The proposed biosensor is fabricated using the cross-linking method to immobilize Alcohol Dehydrogenase (ADH) and Nicotinamide Adenine Dinucleotide (NAD(+)) on the screen-printed electrode modified with Meldola's Blue (MB) absorbed on Nafion. It is based on the electrocatalytic properties of MB as an electron transfer mediator, which can catalyze the oxidation of NADH to NAD(+) at a low oxidizing potential, thus avoiding interferences due to the presence of oxidizable substances in the real serum samples. The biosensor response for alcohol is investigated in terms of pH, buffer solution, temperature and some interferents. It presents the good specificity, reproducibility, stability, accuracy and provides a fast response. The biosensor has been satisfactorily used for the measurement of serum alcohol.

Electrochemical detection of blood alcohol concentration using a disposable biosensor based on screen-printed electrode modified with Nafion and gold nanoparticles.

BACKGROUND: Blood alcohol determination plays an important role in laboratory medicine and forensic medicine. Nowadays, many methods are being used for alcohol measurement, but these methods are time-consuming and complex to perform laborious sample pre-treatment. The disposable amperometric biosensor, due to its portability, low cost and potential for fabrication, should be readily applicable for blood alcohol determination. METHODS: The biosensor was fabricated by immobilizing alcohol dehydrogenase and nicotinamide adenine dinucleotide coated by Nafion combined with gold nanoparticles onto the surface of screen-printed electrode modified with Meldola's blue. Evaluations of biosensor performance were performed according to the relevant National Committee for Clinical Laboratory Standards standard. RESULTS: The biosensor response for serum alcohol presents good linearity, precision, stability, accuracy, and specificity. CONCLUSIONS: The biosensor exhibits the capability of detecting blood alcohol concentration in the clinical laboratory and in forensic medicine, unnecessarily performing laborious sample pre-treatment.