Elemental Labeling Inductively Coupled Plasma Mass Spectrometry Combined With Fluorescence Imaging For Cell Analysis

In the past decades,inductively coupled plasma mass spectrometry(ICP-MS)with elemental labeling strategy has been demonstrated as a revolutionary technique in quantitative bioanalysis with the advantages of high sensitivity,wide dynamic linear range,rapid analysis,good reproducibility,simultaneous detection ability of multi-analytes,and good tolerance to matrix interference;besides,the elemental tags are cheap,stable,biocompatible and easy to obtain.So far,the technology has been applied in the quantification of small organic molecules,biological macromolecules and living organisms.However,there are still some drawbacks in the current technology of ICP-MS with elemental labeling strategy,which limits its application.For example,the function of elemental tags is single,so that in-situ high-resolution images cannot be provided;it is difficult to quantify intracellular biomolecules;relatively large measurement errors exist in conventional measurement methods based on signal intensity of elemental tag.In order to make this technology meet the increasingly stringent demands of current basic biomedical research and clinical applications,appropriate strategies are required to avoid or overcome such defects.The aim of this thesis is to develop new multifunctional elemental tags and design new multifunctional probes to bridge the barrier between ICP-MS-based quantification and optical imaging technology,which would provide more comprehensive information by taking the advantages of different technologies;to reduce the possibility of false-negative or false-positive results,ensuring the accuracy of the proposed method;to develop novel analytical strategies for the quantification and imaging of intracellular micromolecules or macromolecules;and introduce ratiometric measurement methods into ICP-MS-based quantitative bioanalysis to compensate measurement error and suppress signal fluctuation.The major contents of this dissertation are described as follows:(1)A novel magnetic immunoassay based on commercially available CdSe quantum dots(QDs)and Cs-doped multicore magnetic nanoparticles(MMNPs)was developed to achieve ICP-MS ratiometric quantification and fluorescence imaging of HepG2 cells.MMNPs doped with an internal standard(Cs)were prepared and conjugated with anti-EpCAM antibody to prepare capture probes(MMNPs-anti-EpCAM)for the capture of HepG2 cells.CdSe QDs-labeled anti-ASGPR antibody was then used as a signal probe to label HepG2 cells captured by MMNPs-anti-EpCAM specifically.Due to the photoluminescence property of CdSe QDs,fluorescence imaging of HepG2 cells was achieved by confocal laser scanning microscope.At the same time,since CdSe QDs contained large quantities of Cdatoms and MMNPs were doped with Cs,the number of HepG2 cells was counted by ICP-MS detection using the signal ratio of ¹¹⁴Cd/¹³³Cs.The measurement error and signal fluctuation caused by particle loss were compensated and suppressed by this ratiometric quantification method.Under the optimized experimental conditions,the limit of detection(LOD,3?)of the developed method was calculated to be 61 cells with a dynamic linear range of2×10²-3×10⁴ cells,and the relative standard deviation(RSD)for seven replicate determinations of 8×10² HepG2 cells was 5.4%.Then,the proposed method was applied to the determination of HepG2 cells in human blood samples,and good recoveries of 86-104%were obtained.In this method,the counting and fluorescence imaging of cancer cells was achieved with single probe,paving a new way for basic biomedical research and clinical application.(2)A novel multifunctional probe(UCNPs-Ab2-Cy3)was designed to achieve a combination of elemental labeling-based cell counting by ICP-MS with fluorescence imaging and upconversion luminescence imaging.The probe consisted of a recognition unit of goat anti-mouse Ig G to label the mouse anti-EpCAM antibody attached cells,a fluorescent dye(Cy3)moiety for fluorescence imaging as well as an upconversion nanoparticle(UCNP)tag for both ICP-MS quantification and upconversion luminescence imaging of cancer cells.In this method,anti-EpCAM antibody was added to bind to EpCAM overexpressed on the surface of HepG2 cells,and then HepG2 cells bound with mouse anti-EpCAM antibody were specifically labeled with the signal probe UCNPs-Ab2-Cy3.Due to the photoluminescence property of Cy3 and UCNPs,fluorescence imaging and upconversion luminescence imaging of HepG2 cells was achieved by confocal laser scanning microscope.At the same time,since UCNPs contained large quantities of Y atoms,the number of HepG2 cells was counted by the determination of ⁸⁹Y via ICP-MS.Under the optimized experimental conditions,the LOD(3?)of the developed method was calculated to be 1×10² cells with a dynamic linear range of 4×10²-3×10⁴ cells,and the RSD for seven replicate determinations of1×10³ HepG2 cells was 7.1%.The established method provided a proof of concept for the integration of ICP-MS based bioanalysis,together with multimodal bioimaging.In addition,a variety of tags with different functions were integrated into a single probe,giving full play to the advantages of various detection technologies and providing more comprehensive and valuable information.Therefore,it is expected to be a powerful platform for the early and accurate clinical diagnosis of cancer and basic biomedical research.(3)An aptamer-based dual-functional probe(MB-Apt-FAM-AuNP)was designed for rapid and specific ICP-MS-based counting and fluorescence imaging of MCF-7 cell.The probe consisted of magnetic beads for separating and collecting target cells,a recognition unit of aptamer to recognize target cells,a fluorescent dye(FAM)moiety for fluorescence imaging as well as gold nanoparticles(AuNPs)tag for both ICP-MS quantification and fluorescence quenching.Initially,the fluorescence of FAM was quenched by AuNPs.In the presence of target cells,the target cells would bind to the aptamers immobilized on the magnetic beads,and then the AuNPs were released from the probe.The fluorescence of FAM in the probe was recovered due to the release of the fluorescence quencher AuNPs,fluorescence imaging of MCF-7 cells was achieved by confocal laser scanning microscope.In addition,the free AuNPs were rapidly separated from the target cells bound with magnetic beads and the excess unreacted probes by magnetic separation.Based on this,MCF-7 cells were counted by the determination of ¹⁹⁷Auin the supernatant via ICP-MS.Under the optimized experimental conditions,the LOD(3?)of the developed method was calculated to be81 cells with a dynamic linear range of 2×10²-1.2×10⁴ cells,and the RSD for seven replicate determinations of 8×10² HepG2 cells was 5.6%.Then,the proposed method was applied to the determination of MCF-7 cells in human blood samples,and good recoveries of 98-110%were obtained.The method provided a dual-modal detection platform for rapid,simple,sensitive,and specific counting and visualization of MCF-7cell.It is greatly expected as a powerful platform for the rapid and accurate clinical diagnosis of cancer at early stage.(4)A novel dual-functional probe(AgNC@MoS₂)was constructed to achieve a combination of fluorescence imaging and ICP-MS quantification of intracellular ATP.MoS₂ nanosheet was employed as a nanocarrier in the probe,followed by loading a targeted unit of lipoic acid-polyethylene glycol-folic acid and a signal unit of ATP aptamer-templated silver nanoclusters(AgNCs)on the surface.Initially,the fluorescence of AgNCs was quenched by MoS₂ nanosheets.When the probe targeted to the tumor cells by folic acid,the AgNCs were released from the surface of MoS₂nanosheets after binding with intracellular ATP,resulting in fluorescence recovery of AgNCs.The in situ fluorescence imaging of intracellular ATP was achieved by confocal laser scanning microscope.In addition,the excess unreacted probes were removed by centrifugation after the cells were lysed,and the intracellular ATP levels were obtained by the determination of ¹⁰⁷Agin the supernatant via ICP-MS.Under the optimized experimental conditions,the LOD(3?)of the developed method for ATP was calculated to be 0.18 n M with a dynamic linear range of 0.5-2000 n M,and the RSD for seven replicate determinations of 2.0 n M of ATP was 6.7%.Moreover,the obtained results were in good agreement with those obtained by commercial ATP kit via chemiluminescence method,demonstrating good accuracy of the proposed method.Herein,the ICP-MS and fluorescence imaging dual-modal detection of intracellular ATP was achieved with single probe,improving the reliability and simplifying the operation processes.It paves a new way for basic biomedical research,and provides a new strategy for the quantitative analysis of intracellular biomarkers by elemental labeling-based ICP-MS technology.(5)A nanoplatform for cancer therapy and therapeutic self-monitoring was constructed to achieve targeted delivery of anticancer drugs and dual-modal therapeutic self-monitoring by imaging and quantification.Metal organic framework compounds(MOFs)were employed as nanocarriers,followed by loading anticancer drug camptothecine(Cam)in the cavity,along with a targeted unit of folic acid and a signal unit of caspase-3 specific substrate peptide-templated gold nanoclusters(AuNCs)grafted on the surface.Initially,the fluorescence of AuNCs was quenched by MOFs due to photoinduced electron transfer.When the nanocarriers targeted to the tumor cells by folic acid,Cam would release from the cavity of MOFs in the acidic microenvironment of the tumor cells,initiating the apoptosis process and activating caspase-3.Then,AuNCs were released from the surface of MOFs after the substrate peptide linker were cleaved by active caspase-3,resulting in fluorescence recovery of AuNCs.In situ fluorescence imaging of intracellular active caspase-3 was achieved by confocal laser scanning microscope.In addition,the excess nanocarriers were removed by centrifugation after the cells were lysed,and the intracellular active caspase-3 levels were obtained by the determination of ¹⁹⁷Auin the supernatant via ICP-MS.Under the optimized experimental conditions,the LOD(3?)of the developed method for active caspase-3 was calculated to be 0.12 ng m L^-1 with a dynamic linear range of 0.3-150 ng m L^-1,and the RSD for seven replicate determinations of 1.0 ng m L^-1 of active caspase-3 was 6.5%.In this method,the problems of the incapability of common fluorescence imaging techniques to offer quantitative results and the incapability of ICP-MS-based bioanalytical methods to provide real-time and visualized images in previous real-time evaluation of cancer therapeutic efficiency have been solved.Moreover,it provides a new approach to study therapeutic mechanism of cancer.