ß-Cyclodextrin Polymer-Based Fluorescence Enhancement Strategy via Host-Guest Interaction for Sensitive Assay of SARS-CoV-2.

Nucleocapsid protein (N protein) is an appropriate target for early determination of viral antigen-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have found that ß-cyclodextrin polymer (ß-CDP) has shown a significant fluorescence enhancement effect for fluorophore pyrene via host-guest interaction. Herein, we developed a sensitive and selective N protein-sensing method that combined the host-guest interaction fluorescence enhancement strategy with high recognition of aptamer. The DNA aptamer of N protein modified with pyrene at its 3' terminal was designed as the sensing probe. The added exonuclease I (Exo I) could digest the probe, and the obtained free pyrene as a guest could easily enter into the hydrophobic cavity of host ß-CDP, thus inducing outstanding luminescent enhancement. While in the presence of N protein, the probe could combine with it to form a complex owing to the high affinity between the aptamer and the target, which prevented the digestion of Exo I. The steric hindrance of the complex prevented pyrene from entering the cavity of ß-CDP, resulting in a tiny fluorescence change. N protein has been selectively analyzed with a low detection limit (11.27 nM) through the detection of the fluorescence intensity. Moreover, the sensing of spiked N protein from human serum and throat swabs samples of three volunteers has been achieved. These results indicated that our proposed method has broad application prospects for early diagnosis of coronavirus disease 2019.

Use of ß-cyclodextrin-tethered cationic polymer based fluorescence enhancement of pyrene and hybridization chain reaction for the enzyme-free amplified detection of DNA.

Herein, we proposed an enzyme-free strategy for the amplified detection of DNA by combining the efficient fluorescence enhancement capability of a ß-cyclodextrin-tethered cationic polymer (cationic polyß-CD) to pyrene with the amplification capability of target DNA triggered hybridization chain reaction (HCR). Cationic polyß-CD with positive charge was synthesized. Two hairpin probes, H1 and H2, were employed in the system and the pyrene-labelled H2 was chosen as the signal unit. The pyrene attached on the sticky end of H2 was flexible and there was strong electrostatic interaction between cationic polyß-CD and negatively-charged H2, so pyrene could easily enter the cavity of CD that is tethered on the cationic polymer, accompanied by significant fluorescence enhancement. Once target DNA was introduced, HCR was triggered to form a rigid long dsDNA polymer with pyrene attached on it. The pyrene was hardly able to enter the cavity of cationic polyß-CD because of steric hindrance, leading to a weak fluorescent signal. Owing to the efficient pyrene fluorescence enhancement of cationic polyß-CD and the amplified capability of HCR, an enzyme-free sensitive detection of target DNA was achieved with a detection limit of 0.1 nM and high selectivity.

Multiple amplification detection of microRNA based on the host-guest interaction between ß-cyclodextrin polymer and pyrene.

MicroRNAs (miRNAs) participate in various biological processes during the course of life. The levels of miRNAs can be useful biomarkers for cellular events or cancer diagnosis, thus sensitive and accurate analysis of miRNA expression is crucial for better understanding its functions and the early diagnosis of human disease. Here, we developed a multiple amplification detection method for miRNA based on the host-guest interaction between ß-cyclodextrin polymer and pyrene, which takes advantage of the polymerase-aided strand displacement amplification and λ exonuclease-assisted cyclic enzymatic amplification. The proposed method allowed quantitative detection of miRNA-21 in a dynamic range of 1 pM to 5 nM with a detection limit of 0.3 pM and demonstrated good ability to discriminate the target sequence from the single-base mismatched miRNA sequence. Moreover, the assay was applied successfully in a complex biological matrix. We believe that this proposed sensitive and specific assay has great potential as a quantification method for miRNA detection in biomedical research and clinical diagnosis.

A multiple amplification strategy for nucleic acid detection based on host-guest interaction between the ß-cyclodextrin polymer and pyrene.

A multiple amplification strategy has been developed for nucleic acid detection based on host-guest interaction between the ß-cyclodextrin polymer (ß-CDP) and pyrene. Briefly, the detection system consists of three parts: the polymerase and nicking enzyme-assisted isothermal strand displacement amplification (SDA) activated by a target DNA or microRNA; the exonuclease III-aided cyclic enzymatic amplification (CEA); and the fluorescence enhancement effect based on host-guest interaction between ß-CDP and pyrene. This strategy showed a good positive linear correlation with target DNA concentrations in the range from 75 fM to 1 pM with a detection limit of 41 fM. Significantly, our amplification platform was further validated and evaluated successfully by assaying miRNA-21 in human serum. The proposed assay has great potential as a nucleic acid quantification method for use in biomedical research, clinical analysis and disease diagnostics.

[Jingjin needling improves facial nerve function and psychosomatic function in Hunt facial para-lysis patients].

OBJECTIVE: To observe the clinical effectiveness of Jingjin (muscle region) needling in the treatment of Hunt facial paralysis (HFP). METHODS: A total of 80 HFP patients were randomly divided into acupuncture and medication groups (n＝40 cases/group). Jingjin needling was applied to Yangbai(GB14) to Shangxing (GV23), Touwei (ST8), Cuanzhu (BL2), Sizhukong (TE23，penetrative needling), Dicang (ST4) to Jiache (ST6, penetrative needling), Yingxiang(LI20) and Xiaguan(ST7), Hegu(LI4), Yifeng (TE17), Yuyao (EX-HN 4), and Shenmai (BL62), with the needles retained for 30 min. The treatment was conducted once daily, with 10 consecutive days being a therapeutic course, and 3 courses altogether. Patients of the medication group received oral administration of Prednisone acetate (12 days), Acyclovir (7 days), intramuscular injection of Vitamin B12 and Vitamin B1(10 days), then, oral administration of Vitamin B1, successively. The therapeutic effect was assessed by using House-Brackman (H-B) facial function grading system (grade I to VI), traditional Chinese medicine (TCM) sign and symptom score, and facial disability index (FDI) scale including FDI physical function (FDIP, food and water swallowing, speaking-pronouncing, dryness or tearing, and mouth-opening) and FDI social function (FDIS, self-rating anxiety/depression scales), separately. RESULTS: After the treatment, the TCM symptom and sign scores for depth of nasolabial groove, nose shrugging, lower lip asymmetry, food retention and post-auricular pain were significantly lower in the medication group (P<0.05)．After the treatment, the TCM symptom and sign scores for frontal muscle movement, eyelid opening and closing, depth of nasolabial groove, nose shrugging, lower lip asymmetry, cheek blowing, food retention, latissimus cervicalis contraction, taste disorder, hearing hypersensitivity, tears and discomfort and post-auricular pain were significantly lower in the acupuncture group (P<0.05)．Comparison between two groups showed that the TCM symptom and sign scores for frontal muscle movement, eyelid opening and closing, depth of nasolabial groove, nose shrugging, lower lip asymmetry, cheek blowing, food retention, latissimus cervicalis contraction, taste disorder, hearing hypersensitivity, tears and discomfort and post-auricular pain were significantly lower in the acupuncture group than in the medication group(P<0.05)．The scores of FDIP and FDIS were significantly increased in both groups (P<0.01) and notably higher in the acupuncture group than in the medication group (P<0.01) after the treatment. The total effective rate of acupuncture group was 97.5% (39/40), and that of medication group was 72.5% (29/40). The therapeutic effect of the acupuncture group was significantly superior to that of the medication group (P<0.01)．. CONCLUSION: Jingjin needling has a good therapeutic effect in improving facial nerve function, psychosomatic function and clinical signs and symptoms in HFP patients, evidently being better than medication.

N-terminal PEGylation of human serum albumin and investigation of its pharmacokinetics and pulmonary microvascular retention.

Human serum albumin (HSA) is used as an important plasma volume expander in clinical practice. In the present study, HSA was N-terminally PEGylated and a PEGylated HAS (PEG-HSA) carrying one chain of PEG (20 kDa) per HSA molecule was obtained. The purity, secondary structure and hydrodynamic radius of the modified protein were characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis, circular dichroism measurements, and dynamic light scattering, respectively. The pharmacokinetics in normal mice and vascular permeability of the PEG-HSA in a lipopolysaccharide-induced acute lung injury mice model were evaluated. The results showed that the biological half-life of the modified HSA was approximately 2.2 times of that of native HSA, and PEG-HSA had a lower vascular permeability which suggested that PEGylation of HSA could reduce extravasation into interstitial space. It can be inferred that due to the prolonged half-life time and enhanced vascular retention, the molecularly homogeneous PEG-HSA may be a superior candidate as a plasma volume expander in treating capillary permeability increase related illness.