Construction And Bioanalysis Application Of Hemicyanine Near-Infrared Fluorescent Probe

Hemicyanine is a kind of near infrared（NIR）small molecular fluorescent probes with a unique donor-π-acceptor（D-π-A）skeleton,flexible structural tunability and functionally-modified reaction sites.The activable sensing mechanism of the hemicyanine fluorescence probe is mediated by the intramolecular charge transfer（ICT）between the electron donor and the acceptor,thus the hemicyanine scaffold is covalently linked to the biomarker,resulting in ICT blocking and fluorescence signal quenching.Subsequent exposure to the responding target molecule selectively cuts the cage portion,and the fluorescence signal is restored,resulting in"open"or"proportional"fluorescence imaging.By studying the changes of probe fluorescence signals,it is of great significance to reveal the mechanism of action of small molecules or enzymes in cells and their association with diseases,which is of great significance to develop methods of disease diagnosis.Emerging the second near-infrared region（NIR-II,1000-1500 nm）fluorescent probes have greater potential for early prediction and diagnosis of tumors as well as treatment due to their high tissue penetration depth and spatial resolution.However,the emission wavelength of typical hemicyanine is only in the first near infrared region（NIR-I,700-900 nm）,thus how to regulate hemicyanine into the NIR-II fluorescent probe has important research significance.Therefore,this paper mainly focuses on the unique structural advantages and fluorescence properties of hemicyanine and carry out related analysis and research.As well as the challenges faced by hemicyanine,a series of scaffolders were developed and explored to successfully regulate hemicyanine as a NIR-II fluorescent probe,which was applied in biological analysis.The specific work content will be elaborated in six parts.Chapter 1.OverviewIn this chapter,the sensing mechanism of hemicyanine is introduced firstly,and then the research status of hemicyanine structure in recent years is described in detail.Then the strategies and methods for developing NIR-II fluorescent probes and their application advantages are summarized.Finally,the purpose,value and innovative significance of this paper are put forward.Chapter 2.Activated near-infrared fluorescent probes for selective detection and imaging of Hg²⁺in living cells and animalsMercury ion(Hg²⁺)is a kind of harmful heavy metal ion,which has high biological toxicity and serious threat to human health.It can link with mercaptan of enzymes,proteins and even DNA in the biological system,causing serious irreversible damage to the organism.In this chapter,we synthesize a NIR probe（Cy P）based on the hemicyanine structure to achieve the selective detection and imaging of Hg²⁺in living cells and animals.Cy P uses rigid oxanthracene structure as electron donor,trimethylindole as electron acceptor,and diphenylphosphinothioic chloride as recognition group.Cy P could respond specifically to Hg²⁺and turned on fluorescence at 710 nm.In cell and animal imaging experiments,Cy P was specifically activated by Hg²⁺.Meanwhile,Cy P showed high tissue penetration depth and spatial resolution.Therefore,Cy P can be an effective tool for monitoring and evaluating Hg²⁺in living systems.Chapter 3.Tumor-targeting and activatable NIR fluorescent probe detecting for HDAC6 enzyme activityHistone deacetylases（HDACs）are a family of epigenetic enzymes that catalyze the deacetylation of histidine and some proteins.HDACs are considered to be the most promising biomarkers or therapeutic targets.Because they are involved in a number of pathophysiological pathways and diseases,such as cancer,neurodegenerative diseases and immune diseases,it is of great medical and biological value to study intracellular HDACs activity.In this chapter,a NIR fluorescent probe Cy Ac-RGD was synthesized for the detection and imaging of HDAC6 enzymes in cells and tumors.After co-incubation of Cy Ac-RGD with HDAC6,NIR fluorescence singal was turned on.The probe showed high sensitivity and specificity in vitro detection of HDAC6.Moreover,the probe is covalently linked to c-RGD,which can specifically target theα_vβ₃ receptor on the tumor surface to achieve high-resolution,real-time tumor imaging in vivo.Therefore,this chapter develops an optical sensor for detection of HDAC6 to achieve specific detection and imaging of HDAC6 in vitro and in vivo.Chapter 4.p H/viscosity activatable NIR-I/II nanomicelle self-checking for photothermal and photodynamic synergistic therapeutic effectCancer remains a global health challenge,causing millions of deaths each year and thus significantly increasing the public health burden.Assessing the effects of treatment early will greatly benefit cancer patients,saving time for potentially more effective alternative treatments.Conventional tests,however,often take weeks or months after treatment to identify significant tumor shrinkage.Therefore,early and even immediate evaluation of tumor therapeutic effects is of great significance for accurate management of cancer patients.In this chapter,p H and viscosity activatable nanomicelle could timely self-checking the effect of photothermal and photodynamic synergistic therapy.Firstly,we modified the structure of hemicyanine,and HCM with NIR-I/II fluorescent emission was selected as the probe material.In the range of NIR-I region,HCM can respond to changes in viscosity in the environment and has a well photothermal conversion efficiency.Then the probe was assembled into copolymer containing PPa to prepare PPDAM nanomicelles.The nanomicelles can respond specifically to extracellular matrix metalloproteins 2/9（MMP-2/9）.In addition,the nanomicelles dissociated and released HCM probes in tumor environment.PPDAM nanomicelles were observed to be deposited in tumor tissues by NIR-II fluorescence.After 655 nm laser irradiation,PPa turned on the photodynamic therapy and HCM turned on the photothermal therapy,killing the tumor cells.When the tumor cells are in the state of apoptosis,the viscosity of the cells increases and the fluorescence of HCM is enhanced,which can realize the therapeutic effect of timely self-checking of the tumor.Chapter 5.Design of hemicyanine-based NIR-II fluorescent probe for ratiometric monitoring ROS in acute liver injuryDue to the cyanine with more fluorescence brightness compared with hemicyanine,in this chapter,based on the hemicyanine structure,2-methylbenzoindle can react with the C₈-site of HCP to generate cyanine structure DHCP through Michael addition,which significantly improves the fluorescence brightness.DHCP exhibits high sensitivity to reactive oxygen species（ROS）.Thus DHCP and Na Er F₄@Na YF₄nanoparticles were then assembled together with DSPE-PEG₅₀₀₀ and lecithin in a nanomicelle(NPL@DHCP/Er³⁺)to monitor the content of ROS in an acute liver injury model.Therefore,we anticipate that this molecular design strategy provides a new way to construct NIR-II fluorescent probes.Chapter 6.Summary and outlookThis chapter summarizes the work of this paper one by one and further expounds the innovation and research significance of this work.Combined with the current research status and challenges,the research direction was prospected and assumed.