A simple fluorescence assay for trypsin through a protamine-induced carbon quantum dot-quenching aggregation platform

The development of a simple detection strategy for trypsin (Try) is urgent, and is ascribed to the diagnostic value of Try in several diseases. Herein, a facile but effective fluorescence strategy for Try was developed based on the protamine (Pro)-induced aggregation of carbon quantum dots (CQDs). The fluorescence of negatively charged CQDs was quenched with Pro due to the assembly of CQDs and Pro (CQDs/Pro) through electrostatic interaction. However, the highly positively charged Pro, which is rich in basic arginine residues, was preferred to be hydrolyzed by Try. Try can induce the deaggregation of CQDs/Pro, thereby enabling the release of CQDs to restore the fluorescence intensity. Thus, the use of CQDs/Pro as a testing platform will be employed as a “turn-on” method for Try. In addition, the fluorescence-resuming response was proportional to Try, ranging from 25 ng mL⁻¹ to 500 ng mL⁻¹ with a limit of detection (LOD) of 8.08 ng mL⁻¹. This “turn-on” fluorescence assay for Try was label-free, convenient, and relatively free of interference from coexisting substances. Actual applications for Try monitoring and trypsin inhibitor screening also illustrated the considerable prospect of CQDs in the clinical field, combined with the superiority of the simple mixing operation.

In this work, a simple melting method was developed for carbon quantum dot fabrication to integrate with protamine as an effective signal-on fluorescence strategy for trypsin detection.

With increasing attention being paid to human health for disease diagnosis and treatment, increasing efforts have been spent on the development of new analytical methods toward biological macromolecule detection.^1,2 Trypsin (Try) is one of the most vital digestive proteases produced in pancreatic acinar cells, and can catalyze the hydrolysis of peptides mainly found at the C-terminus of lysine and arginine residues.^3–5 Abnormal Try activity will be reflected in pancreatic function and the corresponding pathological changes of the human body. In general, Try is a biomarker for several diseases, such as acute pancreatitis, cystic fibrosis, and pancreatic cancer.^5–7 Therefore, accurate monitoring for Try levels through simple and economical assays has attracted increasing interest in disease diagnosis and treatment.At present, considerable efforts have been devoted to developing efficient and reliable methods for Try detection based on various strategies, including colorimetry,⁸ electrochemistry,⁹ and fluorescence methods.¹⁰ Fluorescent approaches have been widely used due to their characteristics of simplicity, rapidity, sensitivity, and realizing real-time detection.^11,12 Fluorescent methods for Try have mainly been developed based on the hydrolytic effect of Try using peptide chains or specific proteins as substrates,^7,10,13,14 and using fluorescent dots, like CdTe QDs.¹⁴ Although current fluorescent methods are accurate and reliable, a simple and easy sensing system that detects Try with a controllable response integration modulated by Try through a biocompatible probe is still lacking. Given its special structural properties, protamine (Pro) is a highly cationic peptide under physiological conditions that possesses unique physiological function and substrates for enzymatic catalysis.⁸ The arginine-rich structure of protamine can also be catalyzed by Try as an ideal substrate for Try monitoring.^8,15,16 Hence, the interaction system containing Pro and a fluorescence probe, such as AIEgen, can be applied for the analytical assembly of Try.¹⁷ With regard to the relatively complicated preparation of AIE, other fluorescent probes for Try detection are desirable and urgently needed.Among the current fluorescent nanomaterials, fluorescent carbon quantum dots (CQDs) have been used to construct biosensors due to their safety, unique optical properties, and ease of modification.^18–20 Changing the structure or the surface properties of CQDs can result in a difference in the interaction and function between CQDs and external substances.^21,22 Thus, various fluorescent sensors based on CQDs have been established to detect specific targets varying from ions, nucleic acids, and drugs.^22–25 Positively and negatively charged carbon dots with controlled N-doping play opposite adsorption interaction with ssDNA in different applications.^21–23 Hence, a series of different application systems can be proposed to establish by utilizing the different surface properties of CQDs.Herein, negatively charged CQDs, fabricated from a simple melting operation, were applied as the sensing probe for Try detection without any complex surface functionalization or conjugation and compared with other reported quantum dots,²⁶ as shown in Scheme 1. Using Pro as the selective enzymatic substrate for Try, the integration of the prepared CQDs and Pro induce them to assemble and form an aggregation-caused quenching system of CQDs and Pro (CQDs/Pro) via electrostatic interaction. Under the strong and specific hydrolysis of Try to Pro, Pro loses its function with CQDs, and then the CQDs/Pro disaggregates to recover the fluorescence intensity. This facile system using Pro as the medium for inducing CQD aggregation provided novel insights into the design of some quantum dots with the appropriate surface property for the fabrication of simple, selective, and effective fluorescence assays for bioanalysis without a complicated surface functionalization.Open in a separate windowScheme 1The fabrication of CQDs and its electrostatic interaction with protamine as a simple quenching platform for trypsin detection.     

FGF23 Is Endogenously Phosphorylated in Bone Cells

Levels of serum phosphate are controlled by the peptide hormone FGF23, secreted from bone osteocytes. Elevated levels of circulating FGF23 are a key factor in several hypophosphatemic disorders and play a role in chronic kidney disease. Posttranslational processing of FGF23 includes multi‐site O‐glycosylation, which reduces intracellular cleavage by proprotein convertases. The FGF23 protein also contains four serine phosphorylation consensus sequences (S‐X‐D/E); in this work, we asked whether FGF23 is a substrate for secretory phosphorylation. Both HEK cells as well as IDG‐SW3 cells, an osteocyte model, incorporated radiolabeled orthophosphate into intact FGF23, as well as into the 14‐kDa carboxy‐terminal—but not the 17‐kDa N‐terminal—fragment. Sequential serine‐to‐alanine site‐directed mutagenesis of four kinase consensus sites showed that labeling occurred on three serines within the carboxy‐terminal fragment, Ser180 (adjacent to the cleavage site), Ser207, and Ser212. Liquid chromatography‐coupled mass spectroscopy indicated the presence of phosphate at Ser212 in recombinant R&D mouse FGF23^R179Q, confirming labeling results. A phosphopeptide‐specific antibody was raised against phospho‐Ser212 and exhibited immunoreactivity in osteocytes present in mouse long bone, providing further evidence that FGF23 is naturally phosphorylated in bone. Bone SIBLING proteins are serine‐phosphorylated by the ubiquitous Golgi secretory kinase FAM20C. Cotransfection of HEK and MC3T3 cells with FGF23 and active, but not inactive, FAM20C kinase increased the storage and release of FGF23 in radiolabeling experiments, indicating potential effects of phosphorylation on FGF23 stability. Collectively, these data point to an important role for phosphorylation of FGF23 in bone. © 2014 American Society for Bone and Mineral Research.     

外直肌超常量后徙固定在知觉性外斜视手术中的应用

目的 观察外直肌超常量后徙固定在知觉性外斜视手术中的远期疗效,分析其应用价值.方法 回顾性分析我科2012年5月至2013年9月行外直肌超常量后徙固定的知觉性外斜视病例12例.外斜视度数均＞50△,患眼固视功能差.在斜视眼行外直肌超常量后徙固定(缝合于原肌止点后12.0 mm)联合内直肌缩短术(4.0～7.0 mm),观察术后眼位、眼球运动、眼面外观及视功能远期疗效.结果 随访13 ～ 27个月后,12例患者术后眼位正位,眼球外转稍有受限;眼面外观良好;均获得较稳定的患眼固视能力,但未建立明确的双眼视功能.结论 外直肌超常量后徙固定稳定了后徙外直肌的位置,减少了手术肌肉条数,具有安全可行、远期疗效稳定等优点.