Fluorescence Imaging Analysis Of Inflammatory Biomarker

Global epidemiological surveys show the direct relationship between the level of atmospheric particulates,including PM2.5,and the rate of pulmonary dysfunction or respiratory symptom diseases,such as pneumonia.In general,pneumonia is a disease of the alveoli and respiratory bronchioles caused by pathogens,which induce local and systemic inflammatory responses in the patient.Severe complications of pneumonia include meningitis,systemic sepsis,lung abscesses,brain damage,and hearing loss often occur.The mortality rate of patients with pneumonia remains high for the past decades.Clinical diagnosing pneumonia requires multiple techniques and clinical indices,including chest X-ray and CT imaging,culture of blood and expectorated sputum samples,and evaluation of several suggestive clinical features.Chest X-ray and CT imaging play crucial roles in the diagnosis but inflict considerable damages,such as cellular canceration,chromosomal aberration,and germ cell abnormality,which restrict their application.Therefore,effective and rapid imaging approaches must be developed to accurately diagnose pneumonia.Fluorescence imaging method is an ideal method for fluorescence imaging,has caused low damage and has a high resolution advantage of the body.LTA₄H?EC3.3.2.6?is a key protein that mediates the degradation of peptides in host defense mechanism.The expression level of LTA₄H significantly increases during inflammation and is related to the development and progression of inflammation.Therefore,LTA₄H could be an effective biomarker for detecting pneumonia and estimating the effectiveness of the treatment and as a potential molecular target for pneumonia therapy.However,existing detection kits for LTA₄H activity are only applied to the purified LTA₄H in vitro and cannot be used to obtain real physiological data in situ.Moreover,the activity loss of LTA₄H caused by sample preparation may result in severe measurement error.Thus,a fluorescence probe with sensitive and selective response toward LTA₄H must be developed and used to accurately diagnose pneumonia by TP fluorescence imaging of the enzyme activity fluctuations in real-time.For this purpose,we designed and synthesized two fluorescence probes to realize the fluorescence imaging and detection the LTA₄H in cells and mouse model.The two fluorescence probes one is a two-photon fluorescence probe?ASPC?,another is a new type of near-infrared fluorescence probe?Cy-ASP?.The main contents of this paper are as follows:1.We propose a novel two-photon fluorescent probe?ASPC?for detection of LTA₄H activity in vivo.The structure of ASPC contained an amide bond linking the unnatural amino acid L-AspBzl as reaction site and 7-amino-4-methylcoumarin?AMC?as chromophore.LTA₄H could specifically hydrolyze the amide bond between the carboxyl group of L-AspBzl and the amino group in AMC.The activity of LTA₄H in the lung tissues of mice was rapidly and accurately monitored for the first time and could be an indicator for diagnosis of pneumonia.The severity of pneumonia in mice caused by haze particulate was determined through imaging the activity of LTA₄H as biomarker and confirmed using a commercial ELISA kit of interleukin?IL?-1?.This work provides a promising method for clinical detection of pneumonia.2.NIR Fluorescence imaging?FL?has the advantage of high selectivity,high sensitivity,spatial resolution and imaging depth.Therefor,we designed and synthesized a new type of near infrared fluorescence probe Cy-ASP.The probe includes a near-infrared fluorophore?Cy-NH₂?and a specific substrate L-AspBzl amino acid with an amide bond as a recognition site.The probe has near-infrared excitation and emission,enabling long-wavelength imaging of mouse models of pneumonia.This work provides a promising method for clinical detection of pneumonia.